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Authors: Li Kang
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Mice with catheters implanted in the jugular vein (infusions) and carotid artery (sampling) are used for this procedure (V3002). The hyperinsulinemic hypoglycemic clamp involves a constant rate insulin infusion with a fall in blood glucose that is controlled by feed back from regular glucose measurements. Blood glucose is then clamped at a hypoglycemic level. The hypoglycemic clamp is used to test hypoglycemic counterregulation and the functionality of the hypothalamic-pituitary-adrenal axis.

Proper citation: Li Kang 2019. Vandy - Hyperinsulinemic-Hypoglycemic clamp. protocols.io https://dx.doi.org/10.17504/protocols.io.yycfxsw 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. Lipase is a gastrointestinal enzyme that processes fat/lipid digestion.

Proper citation: Jason Kim 2019. U Mass - Lipase. protocols.io https://dx.doi.org/10.17504/protocols.io.x3bfqin Copy   

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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 https://dx.doi.org/10.17504/protocols.io.xzafp2e Copy   

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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 https://dx.doi.org/10.17504/protocols.io.56jg9cn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.yagfsbw Copy   

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Authors: Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: The work of Gordon and colleagues (i.e., Nature. 2006 Dec 21;444(7122):1027-31) has shown that obesity can result in marked shifts in the gut microbiome in mice and other models including humans. While the role of the microbiome remains to be fully elucidated, the gut microbiota can no longer be ignored as a potentially important factor when assessing metabolic phenotype. This service involves 16S gene variable region (V1-V3 or V3-V4 directed primers) sequencing by Titanium 454 (3000 avg seq read depth) of feces, cecal, or other GI contents. Sequences can be processed through a bioinformatics pipeline (Qiime) to taxonomically classify them and to assess alpha and beta diversity of the community. In addition, Principal Components Analysis (PCA) or partial least squares- discriminant analysis (PLS-DA) can leverage variances in the relative microbial abundances to better understand how specific microbes contribute to separation by group. Correlational analyses can identify which variables of host metadata associate with specific microbes. The Core’s gut microbiome assay will employ this approach to uncover unique microbiota fingerprints in test mice. One caveat is that with fecal samples, patterns are only a surrogate for actual gut microbiota patterns, and may not exactly reflect the intestinal populations.

Proper citation: Trina Knotts 2019. UC Davis - Gut Microbiome Analysis (454-3K reads). protocols.io https://dx.doi.org/10.17504/protocols.io.ynkfvcw Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Glucose is oxidized by glucose oxidase to gluconic acid and hydrogen peroxide. The hydrogen peroxide reacts in the presence of peroxidase with HBA and 4-aminoantipyrine forming a red quinoneimine dye. The intensity of the color formed is proportional to the glucose concentration and can be measured photometrically between 460 and 560 nm.

Proper citation: Peter Havel 2019. UC Davis - Glucose Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yj9fur6 Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Lipid metabolism is estimated by measuring systemic clearance of [1-14C] palmitate following a bolus injection in awake mice. Lipid metabolism is altered in obese mice.

Proper citation: Jason Kim 2019. U Mass - Lipid metabolism. protocols.io https://dx.doi.org/10.17504/protocols.io.x3gfqjw Copy   

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Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers
Summary: SummaryA known quantity of tissue / plasma is hydrolyzed and extracted after adding known amounts of internal standards: eg. heptadecanoic acid and cholesterol-d7. Fatty acids / cholesterol are analyzed as their trimethylsilyl derivatives using gas chromatography-electron impact ionization mass spectrometry (GCMS) (note: this protocol outlines the processing for palmitate and cholesterol; other fatty acids and sterols can be assayed using this preparation, see refs 1,2).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. 2009, 284(10):6101-8. 2. Influence of diet on the modeling of adipose tissue triglycerides during growth. Brunengraber DZ, McCabe BJ, Kasumov T, Alexander JC, Chandramouli V, Previs SF. Am J Physiol Endocrinol Metab. 2003, 285(4):E917-25.

Proper citation: Henri Brunengraber 2019. Case - Lipid Analysis Assay by GC-mass spectrometry. protocols.io https://dx.doi.org/10.17504/protocols.io.yehftb6 Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Significant amounts of superoxide dismutase (SOD) in cellular and extracellular environments are crucial for the prevention of diseases linked to oxidative stress. Mutations in SOD account for approximately 20% of familial amyotrophic lateral sclerosis (ALS) cases. SOD also appears to be important in the prevention of other neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's Diseases. The reaction catalyzed by SOD is extremely fast, having a turnover of 2 x 10^6 M-1sec-1 and the presence of sufficient amounts of the enzyme in cells and tissues typically keeps the concentration of superoxide very low. Quantification of SOD activity is therefore essential in order to fully characterize the antioxidant capabilities of a biological system. The Cayman Chemical SOD Assay kit is a fast and reliable assay for the measurement of SOD activity from plasma, serum, tissue homogenates, and cell lysates. SOD activity is assessed by measuring the dismutation of superoxide radicals generated by xanthine oxidase and hypoxanthine in a convenient 96 well format. A key feature of the kit is the inclusion of a quality-controlled SOD standard. The standard curve generated using this enzyme provides a means to accurately quantify the activity of all three types of SOD (Cu/Zn-, Mn-, and Fe-SOD). Each kit contains sufficient reagents to assay 41 samples in duplicate and includes assay buffer, sample buffer, radical detector, SOD (standard), xanthine oxidase, a 96 well plate, and complete instructions.

Proper citation: Peter Havel 2019. UC Davis - Superoxide Dismutase. protocols.io https://dx.doi.org/10.17504/protocols.io.ywdfxa6 Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Cayman's GSH assay kit utilizes a carefully optimized enzymatic recycling method, using glutathione reductase for the quantification of GSH. The sulfhydryl group of GSH reacts with DTNB (5,5'-dithio-bis-2-nitrobenzoic acid, Ellman's reagent) and produces a yellow colored 5-thio-2-nitrobenzoic acid (TNB). The mixed disulfide, GSTNB (between GSH and TNB) that is concomitantly produced, is reduced by glutathione reductase to recycle the GSH and produce more TNB. The rate of TNB production is directly proportional to this recycling reaction which in turn is directly proportional to the concentration of GSH in the sample. Measurement of the absorbance of TNB at 405 or 412 nm provides an accurate estimation of GSH in the sample. GSH is easily oxidized to the disulfide dimer GSSG. Because of the use of glutathione reductase in the Cayman GSH assay kit, both GSH and GSSG are measured and the assay reflects total glutathione. The kit can also be used to measure only GSSG by following an alternative protocol. GSH measurement can be done in plasma, tissue samples, and cultured cells using this kit. Nearly all samples require deproteination before assay.

Proper citation: Peter Havel 2019. UC Davis - Glutathione. protocols.io https://dx.doi.org/10.17504/protocols.io.ykcfusw Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Triglyceride emulsion and heparin will be intravenously infused for 5 hours to acutely raise circulating fatty acids levels in awake mice. Acute lipid infusion is shown to cause insulin resistance in peripheral organs.

Proper citation: Jason Kim 2019. U Mass - Acute lipid infusion. protocols.io https://dx.doi.org/10.17504/protocols.io.xm5fk86 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.

Proper citation: Jason Kim 2019. U Mass - Lactate dehydrogenase. protocols.io https://dx.doi.org/10.17504/protocols.io.x2yfqfw Copy   

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Authors: Chee Lim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Transthoracic mouse echocardiography is used to provide noninvasive imaging of the heart and allows for quantification of myocardialwall and chamber dimensions and systolic and diastolic performance.

Proper citation: Chee Lim 2019. Vandy - Mouse Echocardiography. protocols.io https://dx.doi.org/10.17504/protocols.io.yyifxue Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Insulin clearance test measure systemic clearance of insulin following a bolus injection. Liver accounts for the majority of systemic insulin clearance following secretion from pancreatic β- cells into portal circulation. Hepatic clearance of insulin may be affected by obesity and in other mouse models of altered metabolism. Alterations in insulin clearance may also affect glucose and lipid metabolism.

Proper citation: Jason Kim 2019. U Mass - Insulin clearance. protocols.io https://dx.doi.org/10.17504/protocols.io.x2wfqfe 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 lactate levels are affected by alterations in glucose and protein metabolism.

Proper citation: Jason Kim 2019. U Mass -Lactate. protocols.io https://dx.doi.org/10.17504/protocols.io.x5ufq6w Copy   

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Authors: Douglas J. Rowland
Group: Mouse Metabolic Phenotyping Centers
Summary: The Center for Molecular and Genomic Imaging (CMGI) provides small animal imaging services for preclinical research. Contrast agents are a prerequisite for scientifically useful imaging studies involving microPET and microSPECT. In some studies, a contrast agent is also necessary for microCT imaging studies. Contrast agents include iodine-based compounds for x-ray computed tomography (CT), gadolinium for magnetic resonance imaging (MRI), and radioactive tracers for positron emission tomography (PET) and single photon emission computed tomography (SPECT).

Proper citation: Douglas J. Rowland 2019. UC Davis - Imaging CMGI. protocols.io https://dx.doi.org/10.17504/protocols.io.678hhrw Copy   

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Authors: Amy Ehrlich, Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: Gut tissue (by region- e.g ileum & colon) will be opened along the mesenteric border and mounted in Ussing chambers (Physiologic Instruments, San Diego, CA, USA), exposing 0.3 cm² of tissue surface area to 2.5ml of oxygenated Krebs-glucose (10mM) and Krebs-mannitol (10mM) at 37°C on the serosal and luminal sides, respectively. The paracellular pathway and transcellular pathway will be measured as the flux of FITC-Dextran 4000 (FD-4, Sigma –Aldrich) and horseradish peroxidase (HRP Type VI, Sigma Aldrich), respectively. FD-4 (400µg/ml) and HRP (200µg/ml) will be added to the mucosal chamber and samples will be collected from the serosal chamber every 30 min for 2 hours. Concentration of FD-4 is measured via fluorescence at excitation 485 nm, emission 538 nm. O-dianisidine substrate is used to detect HRP at absorbance 450 nm.

Proper citation: Amy Ehrlich, Trina Knotts 2019. UC Davis - Ex vivo assessment of barrier function-gut permeability. protocols.io https://dx.doi.org/10.17504/protocols.io.yipfudn Copy   

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Authors: Patrick Tso
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Oxygen consumption and carbon dioxide production is measured using indirect calorimetry. The Columbus Instruments Oxymax Equal Flow System is an indirect open circuit calorimeter designed to simultaneously measure metabolic performance of multiple subjects that have similar ventilation needs. uses an open circuit calorimetry technique. This system allows sixteen animal cages to be simultaneously monitored. Variables provided by this measurement include VO2, VCO2, RQ, and HEAT.

Proper citation: Patrick Tso 2019. U Cinn - Energy Expenditure Measurements. protocols.io https://dx.doi.org/10.17504/protocols.io.xi9fkh6 Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:This experiment provides the quantification of multiple hormones using multiplexed-Luminex technology based on beads containing specific antibodies. Sample matrices include plasma, serum, tissue culture supernatants, and tissue or cell lysates. Serum C-peptide levels reflect insulin secretion and pancreatic -cell function. Serum C-peptide levels are altered in obesity, insulin resistance, and type 1 and type 2 diabetes.

Proper citation: Jason Kim 2019. U Mass - C-Peptide. protocols.io https://dx.doi.org/10.17504/protocols.io.xttfnnn Copy   

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