The experiments were supported by the Marga and Walter Boll-Stiftung, Kerpen, Germany; Project 210-02-16 (MAAA), Project 210-03-15 (MAAA) and by the German Research Foundation (DFG; AL1632-02; MAAA), Bonn, Germany; Center of Molecular Medicine Cologne (CMMC; University Hospital Cologne; Career Advancement Program; MAAA), K&#246;ln Fortune (Faculty of Medicine, University of Cologne; KD).

All animal procedures were conducted in compliance with protocols approved by local government authorities (Land NRW, AZ: 2012.A424), and were in accordance with the German animal welfare law and the regulations on the welfare of animals used for experiments or for other scientific purposes. Since lung function analysis may affect lung structure and therefore subsequent histological analyzes, the measurement of Res and Cdyn and the preparation and fixation of the lung for histomorphometry should be performed in different animals. However, measurement of Res and Cdyn following ipGTT is possible. Since stress during the ipGTT could interfere with the anesthesia needed for lung function tests, a recovery period of approximately 2 weeks after ipGTT is recommended to allow mice to recover from body weight loss and changes in blood parameters12.
1. Preparation for Intraperitoneal Glucose Tolerance Test (ipGTT)
NOTE: After 12 h of fasting, the complete ipGTT takes approximately 2 h.
<ol>
	<li>Since stress influences blood glucose significantly, ensure that both adaption of mice, as well as training of the scientist, are performed.</li>
	<li>Transfer the animals to the experimental area under quiet and stress-free conditions.</li>
	<li>Consider the application of a hypercaloric diet to induce obesity in mice. See the discussion section for further advice.</li>
	<li>Fast animals for 12 h overnight, without limiting access to water. The next day, after 12 h of fasting, prepare the blood glucose meter according to the manufacturer&#39;s protocol (see table of materials) by inserting a new test strip into the test strip port.</li>
	<li>Incise the tail tip using sterile scissors, while gently retaining the mouse at its tail, and immediately measure the fasted blood glucose by applying a free-flowing blood drop (minimum sample size 0.5 &#181;L) to the test strip of the blood glucose meter. 
	NOTE: A countdown timer starts on the screen after sufficient application of the blood sample. After 4 s, the test result appears on the screen.</li>
	<li>Afterwards, weigh and label the animals individually using color marking.</li>
	<li>Administer 2 g glucose/kg body weight via intraperitoneal injection. Ensure that the injection volume is 0.1 mL/10 g body weight (27 G needle and 1 cc syringe).</li>
	<li>Subsequently, measure blood glucose after 15, 30, 60, and 120 min by applying a drop of free-flowing blood on a new test strip. 
	NOTE: Blood flow can be increased by gentle massaging of the tail tip-wards. If the tail wound encrusts, clean it using a sterile swab soaked with 0.9% sodium chloride solution.</li>
	<li>Allow the animals to rest in their home cages with unlimited access to water between the measurements.</li>
</ol>
2. Lung Function Analysis to Measure Res and cDyn
NOTE: For undisturbed measurement of Res and cDyn, mice need to be ventilated under deep anesthesia. Stress-free animal handling and proper monitoring of anesthesia are essential. For general instructions using sterile techniques, please review the article by Hoogstraten-Miller et al.15
<ol>
	<li>Calibrate the plethysmograph prior to each set of experiments and prepare the study settings within the software (see Table of Materials).</li>
	<li>Prior to surgery, deeply anesthetize animals via intraperitoneal injection of Xylazine (10 mg/kg body weight) and Ketamine (100 mg/kg body weight) (27 G needle and 1 cc syringe). Ensure that the injection volume is 0.1 mL/10 g per body weight. 
	NOTE: Since ketamine has a proper analgesic effect in mice, no additional pain treatment is necessary. The invasive tracheal catheter/plethysmograph procedure takes approximately 5-7 minutes, then data acquisition can begin.</li>
	<li>Place the mouse in the supine position on a heating pad to maintain the body temperature.</li>
	<li>Cover the eyes with ointment to prevent dryness under anesthesia.</li>
	<li>Constantly monitor the depth of anesthesia using the toe pinch-response. 
	NOTE: Additional administration of anesthetic might be necessary to maintain a surgical plane of anesthesia.</li>
	<li>Moisten the fur of the surgical area in the thyroid region with 70% ethanol.</li>
	<li>Carefully incise the skin in the midline for approximately 1 cm between the jugular notch of the sternum and the tuber symphyses of the mentum by lifting it with forceps and clipping the skin under visual inspection using blunt scissors (Figure 1A).</li>
	<li>Visualize the underlying subcutaneous adipose tissue and thyroid gland.</li>
	<li>Expose the trachea by carefully blunt separating both thyroid lobes at the isthmus and dissection of the sternothyroid and sternothyroid muscles (Figure 1B). Be careful not to harm any vessels and cause bleeding, since this can cause adverse effects on the cardiovascular system and ultimately on the measurements.</li>
	<li>Subsequently, pass a 4-0 braided surgical suture between the trachea and esophagus using blunt forceps. Carefully incise the trachea close to the larynx between the tracheal cartilages with micro scissors.</li>
	<li>Intubate with a tracheal tube (0.04 inch / 1.02 mm diameter) under visual control (Figure 1C). Fix the tube via ligation with the surgical suture to avoid any leak in the system.</li>
	<li>Next, move the animal to the heated bed of the body chamber and connect the tracheal tube to the face plate (Figure 1D) and turn on the ventilation by pressing the ventilation button on the front panel of the controller (Figure 1E).</li>
	<li>Survey the ventilation by observing the thorax movement contemporaneously with the ventilation rate. To confirm proper placement of the tracheal tube, ensure that both sides of the thorax move simultaneously.</li>
	<li>Watch the pressure signal on the computer screen (Figure 1F). Ensure that the ventilation curves are uniform. If this is not the case, detach the animal and check the surgery side. Beware of blood or mucus blocking the tracheal tube. 
	NOTE: For adult animals with a body weight of 20-25 g, the ventilator settings as shown in Figure 2 are suggested in accordance with the manufacturer&#39;s recommendations.</li>
	<li>To control changes in the trans-pulmonary pressure during ventilation, insert an esophageal tube (0.04 inch / 1.02 mm diameter) into the esophagus at the depth that approximates the levels of the lungs. Watch the screen while placing the tube. Place the tube where maximal pressure deflection and minimal heart artifacts can be seen on the screen.</li>
	<li>After surgery, prepare the animal for the measurement. Reinject anesthesia via intraperitoneal injection of Ketamine (100 mg/kg body weight) using a 27-G needle and 1 cc syringe. Ensure that the injection volume is 0.1 mL/10 g per body weight. 
	NOTE: To assess bronchial hyperreagibility, nebulize methacholine, a non-selective muscarinic receptor agonist of the parasympathetic nervous system, which induces bronchoconstriction. Data acquisition is performed in four different phases (Figure 3).</li>
	<li>Start data acquisition according to the manufacturer&#180;s protocol. 
	NOTE: The software automatically guides users through the acquisition process.</li>
	<li>Apply 10 &#181;L of PBS (vehicle) on the nebulizer, and start nebulization after 5 min of acclimation. Next, follow a response phase of 3 min, where Res (cmH2O/mL/s) and cDyn (mL/cmH2O) are measured. At the end, provide a recovery phase of 3 min to the animal prior to the next nebulization.</li>
	<li>Follow the software by stepwise application of 10 &#181;L of increasing concentrations of methacholine (2.5 &#181;g/10 &#181;L, 6.25 &#181;g/10 &#181;L, and 12.5 &#181;g/10 &#181;L) on the ventilator.</li>
	<li>Once all measurements have been performed and recorded, sacrifice the animal by cervical dislocation.</li>
</ol>
3. Lung Isolation for Quantitative Histomorphometric Analysis of Adult Mice
<ol>
	<li>Deeply anesthetize the animal via intraperitoneal injection of Xylazine (10 mg/kg body weight) and Ketamine (100 mg/kg body weight) (27 G needle and 1 cc syringe). The injection volume should be 0.1 mL/10 g per body weight. 
	NOTE: After reaching the state of surgical tolerance, the preparation takes approximately 5 min followed by organ perfusion and 30 min for fixation.</li>
	<li>Once the animal has reached the state of surgical tolerance (negative toe pinch-response), disinfect the animal with 70% ethanol and fix the animal on a pad with surgical tape.</li>
	<li>Sacrifice the animal by cardiac puncture and bleeding. Briefly, open the abdomen with a medial incision through the skin and the peritoneum using blunt scissors.</li>
	<li>Locate the diaphragm head wards of the liver, and carefully separate the liver from the diaphragm.</li>
	<li>Make a small incision in the diaphragm using blunt scissors, and punctate the left ventricle of the heart with a 20 G needle attached to a 2-mL syringe. Slowly exsanguinate the animal. 
	NOTE: Slow and careful exsanguination is important to prevent the ventricles collapsing due to the negative pressure, inhibiting an undisturbed blood flow.</li>
	<li>Dissect the lung by opening the thorax gently through a parasternal incision along the entire length of the rib cage using curved, blunt scissors.</li>
	<li>Afterwards, lift the rib cage to expose the pleural cavity (Figure 3C). Remove the thymus to see the heart and lungs. 
	NOTE: Optional injection of the right ventricle, followed by perfusion of the lung vascular system with ice-cold PBS and then with a fixative solution [e.g., 4% (mass/volume) paraformaldehyde (PFA)] is possible. Be aware that there is an increased risk to rupture alveolar septae and adversely affect lung structure using this method.</li>
	<li>Dissect the lung by first carefully removing the heart.</li>
	<li>Subsequently, pass a 4-0 braided surgical suture between the trachea and esophagus using blunt forceps.</li>
	<li>Next, carefully incise the trachea close to the larynx between the tracheal cartilages, intubate with an intravenous cannula (26 G), and inflate the lung by pressure fixation at a constant pressure of 20 cm H2O using fixative agent [e.g., 4% (mass/volume) of PFA].</li>
	<li>For PFA fixation, leave the fixative for 30 min at room temperature. Afterwards, ligate the trachea and remove the cannula. Then, excise the lung carefully without harming the tissue, and store it in fixative agent at 4 &#176;C overnight. 
	NOTE: Alternatively, according to the ATS/ETS consensus paper 2.5% GA buffered OsO4, Uracil solution is used for proper tissue stabilization. For further tissue preparation, see the consensus paper by Hsia et al.14</li>
</ol>

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R., Limacher, W., Bachofen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+of+rapidly+frozen+lungs:+evaluation+on+the+basis+of+standard+criteria.">Electron microscopy of rapidly frozen lungs: evaluation on the basis of standard criteria.</a> Journal of applied physiology: respiratory, environmental and exercise physiology. 53, 516-527 (1982).</li><li>Rolls, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Process of Fixation and the Nature of Fixatives. , (2017).</li><li>Winsor, L., Woods, A., Ellis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+processing.">Tissue processing.</a> Laboratory histopathology. , 4.2-1-4.2-39 (1994).</li><li>Pearse, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histochemistry, theoretical and applied. , (1980).</li><li>Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+basis+of+alveolar-capillary+gas+exchange.">Morphological basis of alveolar-capillary gas exchange.</a> Physiological reviews. 53, 419-495 (1973).</li><li>Bur, S., Bachofen, H., Gehr, P., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+fixation+by+airway+instillation:+effects+on+capillary+hematocrit.">Lung fixation by airway instillation: effects on capillary hematocrit.</a> Experimental lung research. 9, 57-66 (1985).</li><li>Bachofen, H., Ammann, A., Wangensteen, D., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+fixation+of+lungs+for+structure-function+analysis:+credits+and+limitations.">Perfusion fixation of lungs for structure-function analysis: credits and limitations.</a> Journal of applied physiology: respiratory, environmental and exercise physiology. 53, 528-533 (1982).</li><li>Balcombe, J. P., Barnard, N. D., Sandusky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+routines+cause+animal+stress.">Laboratory routines cause animal stress.</a> Contemporary topics in laboratory animal science. 43, 42-51 (2004).</li></ol>

The authors have nothing to disclose.

This report provides three protocols for three different methods to analyze the impact of obesity on glucose metabolism and pulmonary outcomes. First, the glucose tolerance test offers the opportunity to analyze intracellular glucose uptake and can be indicative of insulin resistance. Second, whole body plethysmography is a technique to measure lung function and is thereby helpful to test the efficacy of novel treatments. Third, a standardized fixation protocol is essential for quantitative morphometric analysis to asses...

According to the World Health Organization (WHO) in 2008, more than 1.4 billion adults, aged 20 and older, were overweight with a body mass index (BMI) greater than or equal to 25; further, over 200 million men and nearly 300 million women were obese (BMI&#8805;30)1. Obesity and metabolic syndrome are major risk factors for a multitude of diseases. While obesity and concomitant increased white adipose tissue mass has been intimately linked to type 2 diabetes2,3, cardio-vascular diseases including coronary heart disease (CHD), heart failure (HF), atrial fibrillation4 and osteoarthritis5, their functional roles in the pathogenesis of respiratory disorders remain poorly understood. However, epidemiological studies have demonstrated that obesity is strongly associated with chronic respiratory conditions, including exertional dyspnea, obstructive sleep apnea syndrome (OSAS), obesity hypoventilation syndrome (OHS), chronic obstructive pulmonary disease (COPD), pulmonary embolism, aspiration pneumonia and bronchial asthma6,7,8,9. Potential mechanisms linking obesity and perturbed metabolism, e.g., insulin resistance and type II diabetes, to the pathogenesis of chronic lung disease not only comprise mechanical and physical consequences of weight gain on ventilation but also induce a chronic subacute inflammatory state10,11. The rise of obesity and lung diseases during the last decade, coupled with the lack of effective preventive strategies and therapeutic approaches, highlights the need to investigate the molecular mechanisms to define new avenues to manage obesity-related lung diseases.
Here, we describe three standard tests, which are important basics to investigate obesity and its impact on lung structure and function in mouse models: (1) intraperitoneal glucose tolerance (ipGTT) (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn); and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. The ipGTT is a robust screening test to measure glucose uptake, and thus the effect of obesity on metabolism. The simplicity of the method allows good standardization, and therefore the comparability of results between laboratories. More sophisticated methods, such as hyperglycemic clamps or studies on isolated islets, can be used for detailed analysis of the metabolic phenotype12. Here we assess glucose tolerance to define an obesity-associated state of systemic and metabolic disorder as the basis for further studies on a pulmonary outcome. To assess the effect of obesity and metabolic disorder on lung function, we measured airway resistance (Res) and respiratory system compliance (Cdyn). To characterize lung disease, unrestrained as well as restrained methods for assessment of lung function are available. Unrestrained plethysmography in freely moving animals mimics a natural state, reflecting breathing patterns; in contrast, invasive methods, such as input impedance measurement of Res and cDyn in deeply anesthetized mice to assess dynamic lung mechanics, are more accurate13. Since chronic respiratory conditions are reflected by histologic alterations of the lung tissue, proper lung fixation for further analysis is imminent. The choice of the method of tissue fixation and preparation depends on the compartment of the lung which will be studied, for example, conducting airways or lung parenchyma14. Here, we describe a method that allows qualitative and quantitative assessment of the conducting airways to study the effect of obesity on asthma development.

<table><tbody><tr><td>GlucoMen LX</td><td>A.Menarini diagnostics, Firneze, Italy</td><td>38969</td><td>blood glucose meter</td></tr><tr><td>GlucoMen LX Sensor</td><td>A.Menarini diagnostics, Firneze, Italy</td><td>39765</td><td>Test stripes</td></tr><tr><td>Glucose 20%</td><td>B. Braun, Melsung, Germany</td><td>2356746</td><td></td></tr><tr><td>FinePointe Software</td><td>DSI, MC s&amp;acute;Hertogenbosch, Netherlands</td><td>601-1831-002</td><td></td></tr><tr><td>FinePointe RC Single Site Mouse Table</td><td>DSI, MC s&amp;acute;Hertogenbosch, Netherlands</td><td>601-1831-001</td><td></td></tr><tr><td>FPRC Controller</td><td>DSI, MC s&amp;acute;Hertogenbosch, Netherlands</td><td>601-1075-001</td><td></td></tr><tr><td>FPRC Aerosol Block</td><td>DSI, MC s&amp;acute;Hertogenbosch, Netherlands</td><td>601-1106-001</td><td></td></tr><tr><td>Aerogen neb head-5.2-4um</td><td>DSI, MC s&amp;acute;Hertogenbosch, Netherlands</td><td>601-2306-001</td><td></td></tr><tr><td>Forceps</td><td>FST, British Columbia, Canada</td><td>11065-07</td><td></td></tr><tr><td>Blunt scissors</td><td>FST, British Columbia, Canada</td><td>14105-12</td><td></td></tr><tr><td>Micro scissors</td><td>FST, British Columbia, Canada</td><td>15000-00</td><td></td></tr><tr><td>Perma-Hand 4-0</td><td>Ethicon, Puerto Rico, USA</td><td>736H</td><td>Surgical suture</td></tr><tr><td>Roti-Histofix 4%</td><td>Roth</td><td>P087.1</td><td>4% Paraformaldehyd</td></tr><tr><td>Ketaset</td><td>Zoetis, Berlin, Germany</td><td>10013389</td><td>Ketamine</td></tr><tr><td>Rompun 2%</td><td>Bayer, Leverkusen, Germany</td><td>770081</td><td>Xylazine</td></tr></tbody></table>

intraperitoneal glucose tolerance test, measurement of lung function, and fixation of the lung to study the impact of obesity and impaired metabolism on pulmonary outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

Representative results of intraperitoneal glucose tolerance test (ipGTT) (Figure 4), lung function test (Figure 5), and representative images illustrating hematoxylin and eosin stained lungs (Figure 6).
The ipGTT was performed in obese mice (blue) after 7 weeks of high-fat-diet (HFD). Standard diet-fed mice served as controls (black). Obese...

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Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

intraperitoneal-glucose-tolerance-test-measurement-lung-function

Research

JoVE Journal

Immunology and Infection

14.1K Views.  University of Cologne. De incidentie van obesitas stijgt en verhoogt het risico van chronische longziekten. De onderliggende mechanismen en preventieve strategieën, welomschreven dier vast te stellen modellen nodig zijn. Hier bieden wij drie methoden (glucose-tolerantie-test, lichaam plethysmography en fixatie van de Long) te bestuderen van het effect van obesitas op pulmonaire resultaten in muizen.

Video: Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Cancer Research

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Medicine

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro, or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis assumes that the metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. This article describes a method for measuring extracellular FD-glucose depletion and validates its correlation with intracellular FD-glucose uptake in cells and tissues ex vivo. Extracellular glucose depletion may be potentially applicable for high-throughput kinetic and dose-dependent studies, as well as identifying compounds with glycemic activity and their tissue-specific effects.

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular depletion of fluorescently labeled glucose correlates with glucose uptake and could be used for high-throughput screening of glucose uptake in excised organs and cell cultures.

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...

Biochemistry

Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical profiles, inclusive of blood glucose levels in both the fed and fasted state, HbA1c, insulin levels, clinical history of diet, body composition, and body weight status, an assessment of normal and abnormal glycemic control can be made. Interpretation of an IVGTT is done through measurement of changes in glucose and insulin levels over time in relation to the dextrose challenge. Critical components to be considered are: peak glucose and insulin levels reached in relation to T0 (end of glucose infusion), the glucose clearance rate K derived from the slope of rapid glucose clearance in the first 20 min (T1 to T20), the time to return to glucose baseline, and the area under the curve (AUC). These IVGTT measures will show characteristic changes as glucose homeostasis moves from a healthy to a diseased metabolic state5. Herein we will describe the characterization of nonhuman primates (Rhesus and Cynomolgus macaques), which are the most relevant animal model of Type II diabetes (T2D) in humans and the IVGTT and clinical profiles of these animals from a lean healthy, to obese dysmetabolic, and T2D state 8, 10, 11.

The goal of this protocol is to present a standard method to perform intravenous glucose tolerance tests (IVGTTs) to assess glycemic control in nonhuman primates and assess their metabolic status from healthy to dysmetabolic.

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical ...

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

Improving Strength, Power, Muscle Aerobic Capacity, and Glucose Tolerance through Short-term Progressive Strength Training Among Elderly People

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in elderly people performing short-term resistance training (RET). Supervised progressive resistance training for 1 h three times a week over 8 weeks was performed by RET participants (71&#177;1 years, range 65-80). Compared to a control group without training, the RET showed improvements on the measures used to indicate strength, power, glucose tolerance, and several parameters of muscle aerobic capacity. Strength training was performed in a gym with only robust fitness equipment. An isokinetic dynamometer for knee extensor strength permitted the measurement of concentric, eccentric, and static strength, which increased for the RET group (8-12% post- versus pre-test). The power (rate of force development, RFD) at the initial 0-30 ms also showed an increase for the RET group (52%). A glucose tolerance test with frequent blood glucose measurements showed improvements only for the RET group in terms of blood glucose values after 2 h (14%) and the area under the curve (21%). The blood lipid profile also improved (8%). From muscle biopsy samples prepared using histochemistry, the amount of fiber type IIa increased, and a trend towards a decrease in IIx in the RET group reflected a change to a more oxidative profile in terms of fiber composition. Western blot (to determine the protein content related to the signaling for muscle protein synthesis) showed a rise of 69% in both Akt and mTOR in the RET group; this also showed an increase in mitochondrial proteins for OXPHOS complex II and citrate synthase (both ~30%) and for complex IV (90%), in only the RET group. We demonstrate that this type of progressive resistance training offers various improvements (e.g., strength, power, aerobic capacity, glucose tolerance, and plasma lipid profile).

The effect of short-term resistance training on elderly people was investigated through the simultaneous use of several methods. Compared to a control group, many improvements were seen, including on muscle aerobic capacity, glucose tolerance, strength, power, and muscle quality (i.e., protein involved in cell signaling and muscle fiber type composition).

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied greatly in small animals, it is difficult to measure the activity of BAT in humans. Therefore, little is known about the heat-generating capacity and physiological significance of BAT in humans, including the degree to which components of the diet can activate BAT. This is due to the limitations in the currently most used method to assess the activation of BAT-radiolabeled glucose (fluorodeoxyglucose or 18FDG) measured by positron emission tomography-computerized tomography (PET-CT).
This method is usually performed in fasted subjects, as feeding induces glucose uptake by the muscles, which can mask the glucose uptake into the BAT. This paper describes a detailed protocol for quantifying total-body human energy expenditure and substrate utilization from BAT thermogenesis by combining indirect calorimetry, infrared thermography, and blood glucose monitoring in carbohydrate-loaded adult males. To characterize the physiological significance of BAT, measures of the impact of BAT activity on human health are critical. We demonstrate a protocol to achieve this by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach will help to understand the physiology and pharmacology of BAT thermogenesis in humans.

Here, we present a protocol to quantify the physiological significance of the impact of brown adipose tissue (BAT) activity on human metabolism. This is achieved by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach can help develop a pharmacological target for BAT thermogenesis in humans.

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

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