The Metabolic Phenotyping Core provides state-of-the-art phenotypical measures to the scientific community at UMMC. Metabolic cages provide accurate measurements of whole metabolic information in rodents. Our metabolic capabilities offer access to Promethion metabolic screening (16 cages available), Columbus Instruments with environmental encloses (8 cages available), and AccuScan Instruments (8 cages) in vivo test to help advance the metabolic characterization of genetic and pharmacological research models of metabolic diseases, circadian rhythms and more. Metabolic cages equipped with oxygen sensors to measure oxygen consumption (VO2) and infrared beams to determine motor activity.
VO2 will be measured for 30 sec-2-min at 10-minute intervals continuously 24-hours a day using an oxygen sensor. Motor activity is determined using infrared light beams mounted in the cages in X, Y and Z axes. Sleep time using duration of immobility of 40s is also available for rodents. The very small movements of the body associated with breathing during sleep are ignored in these measurements.
The XYZ arrays consist of high resolution (1 cm pacing) infrared beam arrays that are invisible to the animal (wavelength ca. 900 nm). The intensity of these beams are measured in rapid succession and movements are easily detected and a centroid-algorithm calculates the animal position and movements. After the mice or rats are acclimatized to the new environment, data are generally recorded for approximately 4-6 days, depending on the needs of the investigators.
In addition, our core performs physiological and non-invasive metabolic experiments to assess insulin sensitivity (glucose tolerance tests, insulin tolerance test, hyperinsulinemic-euglycemic clamp) to assess in vivo insulin action, insulin signaling and glucose metabolism (using labeled isotopes) in awake mice and rats as well as hyperglycemic clamp experiments to assess in vivo pancreatic beta-cell function and its effects on glucose metabolism.
To examine the peripheral disposal of an orally administrated glucose load and to determine the whole body sensitivity of insulin-responsive tissue. For glucose tolerance tests, mice are fasted for 5-6 hours, and glucose will be bolus administered by gavage in awake mice. For insulin tolerance tests, mice are fasted for 5 hrs, and insulin (0.25 or 0.5 U/kg body weight) will be administered (ip or iv) in awake mice. Blood samples will be taken at baseline, 15, 30, 60, 90 and 120 min following glucose administration for measurement of plasma glucose and insulin concentrations.
To determine glucose uptake in different tissues and liver glucose production. Animals will be fasted for 4 hrs, and arterial and venous (femoral) catheters will be implanted under anesthesia for blood sampling and infusion. Then a 24 mCi bolus of [3-3H] glucose will be given i.v. followed by continuous infusion of 0.2 mCi/min infusion for 90 min. After 48 minutes of infusion, a bolus of 50 mCi 2[14C]-deoxyglucose (2-DG) will be given i.v. and blood samples will be taken every 10 minutes from 60 to 90 min and processed to determine plasma [3-3H] and 2DG. At the end of 90 min, animals will be euthanized and heart and skeletal muscle collected and stored at -80oC for glucose uptake and gluconeogenesis analysis performed.
To measure myocyte contractility and myocyte calcium. The heart will be excised, cannulated and connected to a heart perfusion apparatus (Radnoti, CA) and perfusion will initiated in the Langendorff mode. The heart will perfused with a Ca2+- free based. After 3-5 minutes of perfusion, the buffer will be replaced with similar buffer containing collagenase D, collagenase B, and protease type XIV dissolved in 25 ml perfusion buffer.After complete digestion of the heart (6-8 min), the heart will be removed and minced. Extracellular Ca2+ will be gradually added back to the cells. Cardiomyocytes contractility will be assessed using a SoftEdge Myocam system (IonOptix, Westwood, MA). The following parameters will be used to evaluate cardiomyocyte contractility: resting sarcomere length; maximum velocity of shortening (-dL/dt); maximum velocity of re-lengthening (+dL/dt); peak height, maximum change of sarcomere length during contraction; peak shortening, peak height normalized to resting sarcomere length. Intracellular Ca2+ transient will be measured using a dual-excitation, single emission photomultiplier system (IonOptix). Cardiomyocytes will be treated with Fura 2-AM (2 μM) and then exposed to light emitted by a 75 W halogen lamp through either a 340- or 380- nm filter while being stimulated to contract at a frequency of 1 Hz, and fluorescence emissions will be detected. The following parameters will be recorded: calcium baseline signal (F340/380); calcium peak signal, maximum change of calcium signal during contraction (ΔF340/380); % peak calcium change (%ΔF340/380), peak calcium signal normalized to baseline; maximum velocity of calcium change during contraction (+dF/dt); maximum velocity of calcium change during relaxation (-dF/dt).
To measure left ventricular pressure-volume loops to provide a framework for understanding cardiac mechanics in mice and rats. Rats or mice will be anesthetized with isoflurane (1–2 %) and placed on a temperature-controlled heating pad to maintain body temperature. After accessing the internal jugular vein for hypertonic saline infusion using a polyethylene tubing, the right carotid will be carefully dissected, and a catheter (SPR 839 (Mouse), SPR-878 (Rat) Millar Instruments) will be advanced into the left ventricle (LV). Animals will be allowed to stabilize for 5 minutes before transient occlusions of inferior vena cava will be performed to calculate Ees for evaluation of load-independent systolic function, and EDPVRβ will be determined to evaluate end-diastolic stiffness. Ees will be calculated as ESP = Ees × ESV + V0, and EDPVRβ will be calculated as EDP = α × expβ × EDV, where ESP represents end-systolic pressure, ESV represents end-systolic volume, V0 represents calculated pressure when LV volume equals 0, α represents the stiffness and scaling coefficient, and EDV represents end-diastolic volume. To investigate the cardiac responsiveness to volume overload, 12.5 % albumin will be infused at 2 ml/hour. Hypertonic saline (10 % NaCl, 10 μl) will be injected to calculate parallel conductance.
Please consult with the Metabolic Phenotyping Core director prior to submitting the Service Request Form.
Dr. Jussara do CarmoCore DirectorOffice: G261(601)984-4353