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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 dx.doi.org/10.17504/protocols.io.yycfxsw Copy
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 dx.doi.org/10.17504/protocols.io.x3bfqin Copy
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 dx.doi.org/10.17504/protocols.io.ynkfvcw Copy
Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the total concentration of cholesterol present in blood, serum, and plasma. Cholesterol esters are hydrolysed by cholesterol esterase. Cholesterol is then oxidized by cholesterol oxidase with formation of hydrogen peroxide. Peroxidase uses the hydrogen peroxide, phenol, and 4-aminotipyrine to form a quinoneimine dye which is measured at 500 nm.
Proper citation: John Stack, Gary Cline 2019. Yale - Total Cholesterol. protocols.io dx.doi.org/10.17504/protocols.io.y3rfym6 Copy
Authors: Fawaz G. Haj
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
This test is designated to determine if rodents exhibit activation of leptin signaling pathway through evaluation of the phosphorylation state of JAK2 and STAT3 proteins.
Proper citation: Fawaz G. Haj 2019. UC Davis - Leptin Signaling pathway. protocols.io dx.doi.org/10.17504/protocols.io.ybqfsmw Copy
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 dx.doi.org/10.17504/protocols.io.zb4f2qw Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: 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-3one 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 - Total Cholesterol (TC) Protocol. protocols.io dx.doi.org/10.17504/protocols.io.ygeftte Copy
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 dx.doi.org/10.17504/protocols.io.yidfua6 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 II include IL-16 (interleukin-16), IL-17E/IL-25, IL-21, IL-22, IL-28B, EPO (erythropoietin), Exodus-2 (CCL-21), Fractalkine (CX3CL1), MCP-5 (monocyte chemotactic protein-5; CCL-12), MIP-3α (macrophage inflammatory protein 3-alpha; CCL-20), MIP-3β(macrophage inflammatory protein 3-beta; CCL-19), and TARC (thymus and activation- regulated; CCL-17).
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 - Cytokines Panel II - multiplex. protocols.io dx.doi.org/10.17504/protocols.io.xvvfn66 Copy
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 dx.doi.org/10.17504/protocols.io.ykcfusw Copy
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 dx.doi.org/10.17504/protocols.io.ywdfxa6 Copy
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 dx.doi.org/10.17504/protocols.io.xm5fk86 Copy
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 dx.doi.org/10.17504/protocols.io.x2yfqfw Copy
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 dx.doi.org/10.17504/protocols.io.yyifxue Copy
Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers
Summary: This is the standard protocol for routine insulin tolerance testing. It is performed on awake mice, fasted for 18 hr (overnight) or for 6 hours.
Proper citation: Henri Brunengraber 2019. Case - Intraperitoneal Insulin Tolerance Test. protocols.io dx.doi.org/10.17504/protocols.io.yefftbn Copy
Authors: Louise Lantier
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:
The exercise stress test is a maximal exercise test used to calculate VO2 max. Mice are run to complete exhaustion as the treadmill speed is increased every three minutes (See table 1). During the test, direct measurements of the oxygen consumption and carbon dioxide output by the mouse are made, which are then used to calculate VO2 max, VCO2, and RER. VO2 max refers to the maximum amount of oxygen that the mouse can utilize during maximal exercise.
Proper citation: Louise Lantier 2019. Vandy - Exercise Stress Test. protocols.io dx.doi.org/10.17504/protocols.io.yxcfxiw Copy
Authors: Jon Ramsey
Group: Mouse Metabolic Phenotyping Centers
Summary: The major fat pads of mice will be dissected and weighed. This approach provides a way to determine if an intervention has an impact on specific depots of fat.
Proper citation: Jon Ramsey 2019. UC Davis - Adiposity. protocols.io dx.doi.org/10.17504/protocols.io.ybsfsne Copy
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 (10,000 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 (454-10K reads). protocols.io dx.doi.org/10.17504/protocols.io.ykjfuun Copy
Authors: Kristin Evans
Group: Mouse Metabolic Phenotyping Centers
Summary: SummaryA high-fat diet of varying composition and percent fat is administered to induce obesity in mice. High-fat diet induced obesity is causally associated with insulin resistance and type 2 diabetes. Diets will be provided in consultation with the investigator needs. This service applies to specialized feeding for vendor supplied animals being supplied out of the UC Davis vendor approved barrier facility.
Proper citation: Kristin Evans 2019. UC Davis - High fat diet feeding. protocols.io dx.doi.org/10.17504/protocols.io.xfmfjk6 Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of C-reactive peptide reflect systemic inflammation and may be altered in obesity.
Proper citation: Jason Kim 2019. U Mass - C-reactive Peptide. protocols.io dx.doi.org/10.17504/protocols.io.xt5fnq6 Copy
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