Center Overview

The Case Western Reserve University MMPC specializes in metabolic investigations with stable isotopes in vivo and in vitro to characterize mouse models. Our mission is to provide for the scientific community with standardized high quality metabolic and physiologic phenotyping services for mouse models. We are dedicated to assist the investigator in study design and interpretation of their results. One of our key missions is to teach the scientific community metabolism and stable isotope techniques. Our Center offers an annual course “Isotope Tracers in Metabolic Research: Principles and Practice of Kinetic Analysis”. The Center has three cores, Analytical/Metabolomic, Metabolic and Animal Core, to accomplish our mission. The Analytical and Metabolomic Core uses mass spectrometry and labeling patterns to calculate metabolic fluxes such as: lipid, protein and glucose turnover. The Center uses specialized, and in some cases, unique isotopic techniques to investigate the regulation of metabolic pathways and their intracellular compartmentation. The Metabolic Core conducts whole body physiology studies in mice.

Analytical and Metabolomic Core

The Analytical and Metabolomic Core conducts the following types of analyses on tissues, blood, plasma and urine from experiments conducted at the MMPC or at the user's lab. Dr. Puchowicz and her team work out the terms of the contract, help the users understand the nature of the assays, coordinate the planning studies done off-site and the collection and shipment of samples for analyses. The Analytical and Metabolomic Core has a close interaction with the Metabolic Core to perform measurements of glucose, glycerol and/or amino acid turnover that are done in catheterized mice under basal and/or glucose-insulin clamp conditions.

This core provides the following techniques and measurements:

Factors affecting obesity

  • 2H- and 18O-enrichments of plasma or urine water to measure total energy expenditure
  • Turnover of glucose and/or glycerol with [6, 6-2H2]glucose and [2H5]glycerol
  • Rates of FA and cholesterol synthesis in tissues from the incorporation of 2H or 13C

Factors affecting metabolism

  • Concentration and/or labeling pattern of plasma fatty acids (C8-C22) and cholesterol
  • Concentration and/or labeling pattern of plasma aminoacids
  • Concentration and/or labeling pattern of acylcarnitines in plasma or urine
  • Concentration and/or labeling pattern of long-chain acyl-CoAs in tissues
  • Concentration and/or labeling pattern of short- and medium-chain acetyl-CoA, propionyl-CoA, succinyl-CoA, and methylmalonyl-CoA in tissues
  • Concentration and/or labeling pattern of citric acid cycle and intermediates in tissues
  • 13C-labeling pattern of the acetyl moiety of citrate (a proxy of the labeling of mitochondrial acetyl-CoA)
  • Rate of protein synthesis from the incorporation of 2H-enriched water
  • Targeted metabolomic panels in plasma, urine or tissues. Targeted intermediary pathways include those associated with glycolysis, fatty acid synthesis and oxidation and citric acid cycle.

Specialized Metabolism

  • Measurements across organ systems such as liver (via isolated liver perfusions) and brain (blood brain barrier and blood flow analysis, using brain uptake index methods -BUI).

Metabolic Core

The Metabolic Core conducts in vivo and ex vivo metabolic experiments on mice and rats to understand the mechanism for their phenotype. Dr. Croniger assist users in the design and implementation of their study. Dr. Croniger organized approval for their animal protocol with the Case IACUC and plans for the shipment of mice to our center.  She interacts closely with the Animal Care Core for the testing, quarantine, and housing of all mice. If re-derivation of mice shipped to our center is required, the CASE Transgenic and Targeting Core (Dr. Ron Conlon, Director) can provide this service. In vivo imaging by Magnetic Resonance Imaging, XRay, CT, and PET also be conducted by the CASE Imaging Research Core (Dr. Chris Flask, Director).

Case Transgenic and Targeting Facility
Imaging Research Core


This core provides the following techniques and measurements:

Factors affecting obesity and energy balance

  • Diet studies (high fat, Lieber DeCarli ethanol containing diet, special diet) – Administration of diet, Food intake, body weight measurements
  • Continuous monitoring of body temperature, heart rate and/or activity by G2-Mitters®
  • Energy expenditure by indirect calorimetry
  • Exercise studies- treadmill and indirect calorimetry
  • Blood chemistry analysis-metabolic panel
  • Measurement of liver triglycerides

Factors affecting glucose metabolism

  • Glucose Tolerance Test (GTT)
  • Insulin Tolerance Test (ITT)
  • Portal vein injection of insulin and tissue collection (basal and 5min after insulin injection)
  • Hyperinsulinemic euglycemic glucose clamp
  • β-islet isolation and culture

Factors affecting liver metabolism

  • Bile duct ligation
  • 2/3 partial hepatectomy
  • Lieber DeCarli ethanol containing diet administration
  • Alanine aminotransferase concentration (ALT)

Surgery and tissue collection

  • Chronic or acute arterial and/or jugular catheterization
  • Liver perfusion and heart perfusion
  • Tissue perfusion and fixation

Service Pricing

Price Table

Test Descriptions

CA2000:  Body Composition using :2H2-labeled water

A bolus of labeled water, 2H2O, is administered by intraperitoneal injection to fasted mice. After allowing for isotope equilibration (2 h), a blood sample is collected by the tail vein, and the 2H enrichment of body water is measured using gas chromatography-mass spectrometry. Body water is calculated from the isotope dilution, and the fat mass is determined


CA2001:  Food Consumption

Food consumption is measured over a 24-hour period. This is done once a week for the duration of the study.


CA2002:  Body Weight

Mice are weighed 1-3 times per week for the duration of the study.


CA2003:  Continuous measurement of body temperature (by probe)

Body temperature will be measured by anal probe for a short period of time. This can be done at room temperature or under cold challenge conditions.


CA2004:  GTT - Glucose Tolerance Test

Mice will be fasted for 5 hours. A fasting blood sample will be removed from the tail vein and a concentrated solution of glucose injected into the abdominal cavity of the mice through a needle passed through the abdominal skin.  Blood samples (~ 5ul) will be removed from the tail vein 15, 30, 60 and 120 minutes later and glucose will be measured by glucometer. Blood samples may also be taken for measurement of insulin by ELISA.


CA2005: Insulin concentrations at fasting and post intraperitoneal glucose

administration Mice will be fasted for 5 hours. A fasting blood sample will be removed from the tail vein and a concentrated solution of glucose (2gm/kg body weight) injected into the abdominal cavity of the mice through a needle passed through the abdominal skin.  Thirty minutes later, another blood sample will be taken.  Insulin in plasma at time 0 and 30 min. will be measured by ELISA. (CA2006)


CA2006:  Plasma insulin measurement by ELISA 

We will measure insulin concentration in plasma from mice (~25ul/mouse in duplicate) by commercially available ELISA.  


CA2008:  Insulin Tolerance Test (ITT)

Glucose concentrations at fasting and post intraperitoneal insulin administration. Mice will be fasted for 5 hours and anesthetized.  A fasting blood sample will be removed from the tail vein and insulin (0.5mU/g) will be injected into the abdominal cavity. Blood samples will be removed from the tail vein 15, 30, 45 and 60 minutes later and glucose will be measured by glucometer.


CA2009:  Triglycerides in liver

A small piece of liver tissue will be homogenized and liver triglycerides will be saponified in KOH. Glycerol will be measured against glycerol standards using a commercially available reagent set.


CA2010:  Plasma Triglycerides

Plasma triglycerides will be saponified in KOH. Glycerol will be measured against glycerol standards using a commercially available reagent set.


CA2011:  Total Energy Expenditure using (2H, 18O-labeled water)

TEE is equal to the sum of basal metabolic metabolic, thermic effect of eating and physical activity. Following a single bolus injection of 2H and 18O-labeled water one can estimate TEE via the elimination rates of 2H and 18O from body water. This test requires serial measurements of the labeling of body water over approximately 1 week, which necessitates the collection of blood or urine samples daily. Note: This test does not require catheterized mice, nor does it require that mice be shipped to the MMPC. The isotopes are non-radioactive and no special safety precautions are required, the tracers will be shipped from the MMPC to the investigator. Investigators will be instructed on how to administer the isotopes, collect samples and then ship them back to the MMPC.


CA2013:  Hyperinsulinemic Clamps (Hypoglycemic or Euglycemic)

The euglycemic-hyperinsulinemic clamp is used to investigate insulin action and glucose metabolism in the intact, conscious mouse. The use of different glucose or glucose analog tracers with the option of using radio-labeled (3H or 14C labeled) or stable-labeled (2H or 13C labeled) allows the determination of tissue-specific glucose uptake in addition to whole body glucose production and disposal. A chronic in-dwelling catheter is surgically implanted into the right jugular vein of the mouse.  After a minimum of 4 days of recovery, the mice are fasted the morning of the experiment.  The 120-minute protocol begins with a prime-continuous infusion of glucose tracer and a prime- continuous infusion of insulin, at a rate dependent on the goals and conditions of the experiment.  Glucose is infused at a rate that maintains euglycemia and is a measure of insulin sensitivity.  Thirty minutes before the end of the study 2-deoxyglucose is administered and tissues will be collected at the conclusion of the clamp for later assessment of tissue-specific glucose uptake as well as other biochemical assays as desired. CA2015: Turnover of glucose, lipid and/or protein During a constant tracer infusion, the dilution of the infused tracer yields a measure of that molecule's rate of appearance. One can measure the turnover of numerous molecules using this strategy. One can determine the kinetics glucose, glycerol and protein using [6,6-2H2]glucose, [2H5]glycerol and [2H5]phenylalanine.  Note: This test requires a catheterized animal.


CA2016:  Fatty acid and cholesterol synthesis using 2H-labeled water

Rates of fatty acid and cholesterol synthesis can be determined in tissues via the incorporation of 2H into lipid intermediates. For example, following a bolus injection of 2H-labeled water one can collect samples (e.g. blood, liver and/or adipose tissue). The respective lipids are isolated and their 2H-labeling is determined. This test can be performed in 2 modes, short term vs long term. In a short term study, the tracer is administered and samples are collected within hours to determine the synthesis of lipids in plasma and/or liver. In a long term study, the tracer is continuously administered over several days.  Samples of adipose tissue are collected. The difference in time scale is necessary since the pool of lipids in adipose tissue is relatively large and requires more time for label to appear. Note: This test does not require catheterized mice, nor does it require that mice be shipped to the MMPC. The isotopes are non-radioactive and no special safety precautions are required, the tracers will be shipped from the MMPC to the investigator. Investigators will be instructed on how to administer the isotopes, collect samples and then ship them back to the MMPC.


CA2017:  Tissue-specific protein synthesis using 2H-labeled water

Rates of protein synthesis can be determined from the incorporation of 2H-labeled water into proteins. For example, following a bolus injection of 2H-labeled water one collects samples (e.g. blood, liver, muscle, etc). 2H-labeled amino acids are then assayed by GC-MS to measure total protein fractional synthesis rates in tissues. This test can be performed in 2 modes, short term vs long term. In a short term study, the 2-H tracer is administered and samples are collected within hours to determine the synthesis of proteins in plasma, liver, etc.   This mode is well-suited for examining the acute response of protein synthesis to a perturbation (e.g. food intake).  In a long-term study, the tracer is continuously administered over several days.  Samples are collected and the assays are performed. The long term design yields an integrative measure of protein synthesis;  i.e.: the isotope is present during the fed and the fasted state and accounts for all protein synthesis over such a transition.  Note: This test does not require catheterized mice, nor does it require that mice be shipped to the MMPC. The isotopes are non-radioactive and no special safety precautions are required, the tracers will be shipped from the MMPC to the investigator. Investigators will be instructed on how to administer the isotopes, collect samples and then ship them back to the MMPC.


CA2018:  Profile of acylcarnitines in plasma/urine or tissue samples

These LC-MS/MS assays are routinely performed to assess changes in acylcarnitine concentrations and profiles as a result of modifications in mitochondrial energy metabolism. This testcoupled with the assay of the profile of urinary organic acids helps in the characterization of a number of metabolic defects, such as inborn errors of fatty acid oxidation disorders.


CA2019:  Profile of long chain acyl-CoAs in tissue

Commercial preparations of CoA and acyl-CoA contain an unnatural analog of CoA, iso-CoA, in which the 3' phosphate has been moved to the 2' position of ribose. We can use the acyl-iso-CoA esters as internal standards to calculate the concentration and mass isotopomer distribution of acyl-CoAs from LC-MS data.


CA2020:  Measurement of acetyl-CoA, propionyl-CoA and/or succinyl-CoA in tissue

Commercial preparations of CoA and acyl-CoA contain an unnatural analog of CoA, iso-CoA, in which the 3' phosphate has been moved to the 2' position of ribose. We can use the acyl-iso-CoA esters as internal standards to calculate the concentration and mass isotopomer distribution of acyl-CoAs from LC-MS data.


CA2022:  13C-Labeling pattern of acetyl moiety of citrate (substrate oxidation)

A number of investigators, who use 13C-labeled precursors of acetyl-CoA -in vivo- in isolated organs or in cell incubations, have attempted to estimate the labeling of mitochondrial acetyl- CoA to calculate the contribution of the substrate to the acetyl-CoA oxidized in the citric acid cycle (CAC). The best proxy for mitochondrial acetyl-CoA is the acetyl moiety of citrate. We developed an assay of the labeling of the acetyl moiety of citrate which involves (i) tissue extraction, (ii) alkaline hydrolysis of extant acetyl-CoA, (iii) after pH adjustment, cleavage of citrate with CoA + ATP-citrate lyase which we isolated from rat liver. The acetyl-CoA formed is either assayed as such by LC-MS, or reacted with thiophenol, followed by GC-MS assay of acetylthiophenol. This assay allows one to calculate the contribution of two or three substrates to mitochondrial acetyl-CoA in the same experiment. For example, consider a mouse heart perfused with unlabeled glucose + [1-13C]palmitate + [U- 13C4]acetoacetate. These substrates yield acetyl-CoA that is unlabeled (M), singly labeled (M1), or doubly labeled (M2), respectively.  So the percent abundances of the M, M1, and M2 mass isotopomers of the acetyl moiety of citrate yield the contribution of each of the substrates to mitochondrial energy production.  


CA2024:  Metabolomic profile of citric acid cycle and gluconeogenic intermediates

We assay the relative concentration of citric acid cycle intermediates and those of the gluconeognic pathway. Assays are run using samples from mice tissues/organs that have also been infused with a 13C-labeled tracer, e.g. 13C-lactate.  This strategy allows one to determine flux rates (via the 13C-labeling patterns) and identify points of control of a pathway, e.g. gluconeogenesis (via the relative concentration and labeling profiles).


CA2024CT:  CUSTOM-DESIGNED TRACER EXPERIMENT

We help users design experiments aimed at measuring: (i) carbon flux through a pathway, or  (ii) the distribution of carbon fluxes through multiple pathways. Since our analytical approach is mass spectrometry based (GC-MS, LC-MS/MS), most of the metabolite analysis listed can also include isotopomer analysis when a stable isotope is given / applied. We emphasize the need to distinguish net carbon flux from isotopic flux. The latter often results from net carbon flux, isotopic exchange, or both. We suggest the use of isotopic substrate(s) and analytical equipment (mass spectrometry, NMR) most appropriate to the problem being studied. If the isotopic substrate is not commercially available, we can, in some cases, arrange for a pilot synthesis of the compound in an academic lab.  We welcome users and their staff to spend time in our lab to learn and practice isotopic techniques.  (INQUIRE with Dr. Brunengraber  or  Dr. Puchowicz directly, if necessary)


CA2025:  Chronic arterial and jugular vein catherization

Mice are anesthetized with isoflurane. Under sterile surgical conditions, permanent catheters are inserted into one jugular vein and one carotid artery. The catheters are funneled to the back of the neck, filled with heparinized saline and plugged. The skin is sutured. The mice are allowed to recover for 2-4 days before being used for metabolic studies if they have recovered their pre- surgical body weight. The patency of the catheters is periodically checked, by flushing with heparinized saline.


CA2026:  Chronic arterial or jugular vein catherization

Mice are anesthetized with isoflurane. Under sterile surgical conditions, a permanent catheter is inserted into one jugular vein or one carotid artery. The catheter is funneled to the back of the neck, filled with heparinized saline and plugged. The skin is sutured. The mice are allowed to recover for 2-4 days before being used for metabolic studies if they have recovered their pre- surgical body weight. The patency of the catheter is periodically checked, by flushing with heparinized saline.


CA2027:  Acute arterial and jugular vein catherization

Mice are anesthetized with isoflurane. Under sterile surgical conditions, permanent catheters are inserted into one jugular vein and one carotid artery. After 30 min of recovery, during which rectal temperature is monitored and maintained at 37ºC, the catheters are connected to syringe pumps or syringes and the acute metabolic investigation is started. At the end of the experiment, the mouse is euthanized by a bolus injection of pentobarbital.


CA2028:  Acute arterial or jugular vein catherization

Mice are anesthetized with isoflurane. Under sterile surgical conditions, a permanent catheter is inserted into one jugular vein or one carotid artery. After 30 min of recovery, during which rectal temperature is monitored and maintained at 37ºC, the catheter is connected to a syringe pump or a syringe and the acute metabolic investigation is started. At the end of the experiment, the mouse is euthanized by a bolus injection of pentobarbital. 


CA2029:  Acute portal vein catherization

Mice are anesthetized with isoflurane. Under sterile surgical conditions, a permanent catheter is inserted into the portal vein through mid-line laparotomy. The skin is sutured. After 30 min of recovery, during which rectal temperature is monitored and maintained at 37ºC, the catheter is connected to a syringe pump or a syringe and the acute metabolic investigation is started. At the end of the experiment, the mouse is euthanized by a bolus injection of pentobarbital. 


CA2030:  Implant [G2 E – Mitters™]

An E-Mitter is a small implantable mouse telemetry system that is powered by capturing energy from electrical fields generated by the ER-4000 Energizer/Receiver. This allows the E-Mitter to operate without batteries and remain implanted indefinitely to monitor the subject's temperature,gross motor activity or heart rate data. Adult mice will be anesthetized and the E-Mitter telemetry device (available from Star Life Science) will be placed under sterile conditions in the abdomen.


CA2031:  Long-term analysis of surgery implantation on G2 E-Mitter™

Animals will be allowed to recover from implanting the E-Mitters for at least seven days before the onset of testing. The receiver device will measure physiological variables such as heart rate, body temperature and activity. These measurement can be used for several days to a week or throughout a diet study.


CA2040:  Targeted metabolomic profiles of Free or Total Fatty Acids/Sterols in plasma, urine or tissue

Concentrations of fatty acids and sterols can be quantified using added internal standards to the samples. Changes in profiles can be used to assess alterations in lipid and sterol metabolism that is often associated with insulin resistance and obesity.


CA2041:  Tissue processing by Pathology Core

Animals will be anesthetized and tissues will be harvested as requested. These tissue will be frozen in liquid nitrogen or fixed. The tissues will be processed by Case Pathology Core as requested by the investigator.


CA2043:  Portal vein injection

To observe the effects in insulin signaling, insulin (1 milli-unit · kg-1 body weight) will be administered to anesthetize mice by injection into the portal vein. Liver, skeletal muscle and fat biopsies will be removed before basal-time (0) and 5 min after the insulin injection. The tissues will be quick-frozen for the investigator's future analysis, using the Western Blot Procedure.


CA2044:  Brain uptake and blood flow

Brain uptake and blood flow are simultaneously measured, in vivo, using the single pass dual label tracer indicator method, in vivo (modified brain uptake index method).


CA2045:  Measurement of ATP/ADP

In mice, ATP tissue concentrations are measured in BAT and WAT/tissues. Samples are dissected from mice under anesthesia and flash-frozen. ATP tissue concentrations are analyzed enzymatically from 25-30 mg of tissue


CA2046:  Indirect Calorimetry

Metabolic rates are measured by indirect calorimetry in mice using an 8-chamber open-circuit Oxymax system (CLAMS, Columbus Instruments, Columbus, OH). Briefly, mice are acclimated to the experimental room for 1 week prior to the experiment. The mice will be individually housed in acrylic calorimeter chambers through which air with a known O2 concentration is passed at a constant flow rate. The system automatically withdraws gas samples from each chamber hourly for 24 h. The system then calculates the volumes of O2 consumed (VO2) and CO2 generated (VCO2) by each mouse in 1 h. The RQ, which is the ratio of VCO2 to VO2, is calculated. Heat or energy expenditures are measured throughout the study measurements are carried out in both light and dark cycles and both fed and fasting conditions and are represented as Kcal/g/day. Mice are maintained at 25°C and had free access to water in all conditions.


CA2047:  Treadmill Training/Endurance Study PLUS Indirect Calorimetry (for 8 mice)

Resting measurements were performed when the mice were noted to be quietly resting and not sleeping or engaging in grooming or feeding activities. Metabolic measurements during exercise will be performed utilizing a Simplex II metabolic rodent treadmill (Columbus Instruments) that allow volumetric gas analysis. Adjustable parameters on the treadmill include belt speed (O-99 m/min) and angle of inclination (O-45”). After acclimation for 1 h and after stable baseline VO2, VCO2, and RER measurements were recorded, 2.5m/min incremental increases in treadmill belt speed and 2’ increments in angle of inclination were performed every 3 min until the mouse exhibits signs of exhaustion. Exhaustion is defined as the mouse spending >50% of the time or X 5 s consecutively on the shock grid. The maximum speed and duration of the run was recorded at the time of exhaustion. A blood sample is taken before and after exercise for determination of lactate using an autoanlyzer (YSI 2700 Biochemistry Analyzer).


CA2051:  Excise Tissues, Blood Serum/Plasma

Animals will be anesthetized and blood and tissues will be harvested as requested. These tissue will be frozen in liquid nitrogen or fixe


CA2052:  Isolated mouse HEART perfusion

Isolated mouse HEART perfusion.


CA2053:  Isolated mouse LIVER perfusion

Isolated mouse LIVER perfusion. 


CA2053CB:  CUSTOM-DESIGNED BIOLOGICAL EXPERIMENT

We help users to design and implement in vivo experiments aimed at unraveling the metabolic process they are investigating. Protocols use a combination of physiological, metabolic and, if needed, isotopic techniques. The experiments and/or analyses are conducted in the user's lab or at the MMPC, with some of the analyses conducted in one of the MMPCs.

Meet The Team

Henri Brunengraber

Center Director

(216)368-6429

hxb8@case.edu

John Kirwan

Center Co-Director

(216)444-3412

kirwanj@ccf.org

Michelle Puchowicz

Analytical Core Director

(216)368-2501

map10@case.edu

Colleen Croniger

Metabolic Core Director

(216)368-4967

cmc6@case.edu

Contact Info

Case MMPC
The CASE MMPC
Case Western Reserve University
10900 Euclid Ave., BRB 9th Floor
Cleveland, OH 44106-4988
P: (216) 368-4268