This work was supported by a National Imaging Facility Subsidised Access Grant to BJC, a National Health and Medical Research Council of Australia program grant (482800) to KAR and PJB. The authors would like to thank Andrew Arthur, Hasar Hazme and Marie-Claude Gregoire for support in developing this method.

All procedures described in this study were approved by the Sydney Local Health District and University of Sydney Animal Ethics Committees and followed the NIH Guide for the care and use of laboratory animals, Eighth edition (2011).
1. Animal Preparation
NOTE: In this protocol male db/db mice (BKS.Cg-Dock7m +/+ Leprdb/J) were maintained in group housing with ad libitum access to chow and water until 6 weeks of age. At the time of imaging, mice weighed ~ 30 g. All mice used in this protocol had fasting blood glucose levels between 10 and 14 mmol/L.
<ol>
	<li>If required, fast the mice. In the present example, fast the mice for 5 h prior to the experimental procedure.</li>
	<li>Treat mice with the desired agent (e.g. drug, protein, peptide) before the commencement of imaging. In this example, administer a subcutaneous injection of insulin (3 U/kg human insulin) or equivalent volume PBS 30 min prior to the start of imaging.</li>
</ol>
2. Set up Workflow
NOTE: This protocol was implemented on a PET/CT scanner. Acquire PET data first, followed by acquisition of CT data.
<ol>
	<li>PET settings:
	<ol>
		<li>Select isotope as 18F, set scan duration to 3,600 s, and upper and lower level energy discrimination to 350 keV - 650 keV (default) with a coincidence timing window of 3.432 ns (default). Histogram list-mode data into 16 frames (6 &#215; 10 s, 4 &#215; 60 s, 1 &#215; 300 s, 5 &#215; 600 s) for the period 0 - 60 min after tracer injection. Reconstruct emission sinograms using 2D-FBP with a zoom 1.5. 
		NOTE: The reconstructed images consisted of 16 dynamic frames, each with 128 &#215; 128 &#215; 159 voxels and a voxel size of 0.52 &#215; 0.52 &#215; 0.796 mm3.</li>
	</ol>
	</li>
	<li>CT settings:
	<ol>
		<li>For a whole body CT scan, set the current at 500 A, voltage at 50 kV, exposure time 500 ms and 200 projections over a 360 rotation. Set the detector field of view (FoV) to 30722048, number of bed positions to 3 (to cover full PET FoV range), overlap between bed positions = 30.234713% and detector binning to 4. 
		NOTE: CT reconstruction was performed using cone beam tomography image reconstruction software with HU calibration, bilinear interpolation and Shepp-Logan filter.</li>
	</ol>
	</li>
	<li>18 F-FDG:
	<ol>
		<li>Order sufficient 18F-FDG (for example 450 MBq in 0.5 mL) from a local provider to arrive ~ 30 min before the first injection. Aliquot and dilute the 18F-FDG so that animals receive ~ 10 MBq of 18F-FDG in a final volume of 0.1 mL.</li>
	</ol>
	</li>
</ol>
3. Imaging Protocol
<ol>
	<li>Wipe down induction chamber and imaging bed with 80% (v/v) ethanol to maintain aseptic conditions. Place the mouse in an induction chamber and anesthetize with 5% isoflurane in oxygen.</li>
	<li>Place the mouse onto an imaging bed fitted with an electric heating pad to maintain body temperature and a precision vaporizer nose cone to deliver isoflurane (maintenance, 1.5 - 2%) at a flow rate of 1 L/min. Apply ophthalmic ointment onto eyes to prevent dryness whilst under anesthesia.</li>
	<li>Position the mouse in a prone position on a sensor pad to monitor breathing and ensure an adequate plane of anesthesia is maintained.</li>
	<li>Warm the tail using a heat pack for 1 - 2 min to dilate the lateral tail vein. Catheterize the lateral tail vein by inserting a 30-gauge needle into the lateral tail vein. Secure the needle in place with surgical glue and secure the catheter.</li>
	<li>Load imaging bed into the scanner and move the bed through the machine so that the catheter can be accessed from the rear of the machine.</li>
	<li>Attach the catheter to the 18F-FDG syringe in a syringe driver. Calculate the exact 18F-FDG dose (10 MBq) based on activity in syringe before injection and the volume to be administered (&#60; 100 &#956;L, injected over 10 s).</li>
	<li>To minimize the impact of anesthesia on variability of glucose uptake, ensure a constant time between induction of anesthesia and injection of 18F-FDG (for example, 30 min).</li>
	<li>Commence the PET scan immediately before injection of 18F-FDG. After finishing the PET scan (3,600 s), perform a CT scan (~ 10 min) to allow for co-registration of radiotracer uptake with tissues.</li>
	<li>Move the imaging bed to the starting position, remove the animal from the bed.</li>
	<li>At this point euthanize the animal or allow it to recover:
	<ol>
		<li>For euthanasia, perform cervical dislocation whilst still under anesthesia and collect organs of interest for subsequent analysis.</li>
		<li>If allowing the mouse to recover, place the mouse in single housing on a heating pad or in front of a heating lamp. Monitor the mouse until it has regained sufficient consciousness to maintain sternal recumbency. Allow the mouse to recover for 1 h before returning to group housing.</li>
	</ol>
	</li>
</ol>
4. PET Image Processing
NOTE: Image reconstruction was performed using acquisition workplace software v1.5.0.28 and analysis in research workplace software v4.2.
<ol>
	<li>Co-register CT and PET images and ensure that the alignment is correct in all 3 dimensions.
	<ol>
		<li>In the &#39;File&#39; menu, select &#39;Folder Search/Import&#39; and select the folder containing the data. Select the desired PET and CT data and click on the &#39;General Analysis&#39; tab.</li>
		<li>Sort the data so that the CR is designated &#39;Source&#39; and PET is designated &#39;Target&#39;. In the &#39;Workflow&#39; menu, select &#39;Registration&#39;. If the images require adjustment to be correctly co-registered, use the tools in the &#39;Registration&#39; menu.</li>
	</ol>
	</li>
	<li>In the &#39;Workflow&#39; menu, select &#39;ROI Quantification&#39;.
	<ol>
		<li>Use the &#39;Pan&#39; and &#39;Zoom&#39; functions in the &#39;Image&#39; tab to locate the desired region. In the &#39;Tools&#39; menu, select the &#39;Create&#39; tab and click the paintbrush icon. Draw the ROI on the image</li>
	</ol>
	</li>
	<li>Extract time-activity curves by selecting &#39;Save ROI Quantification&#39; from the &#39;Save&#39; menu. Save the data as a CSV file.</li>
	<li>Quantify radioactivity uptake as Bq per cm3 of tissue. Convert values into percentage of injected dose per cm3 (% ID/ cm3) by loading the CSV file into a spreadsheet.</li>
</ol>
5. Input Function
<ol>
	<li>To correct for the system point spread function, deconvolve the estimated system PSF for 5 iterations using the reblurred Van Cittert deconvolution method as previously described9. 
	NOTE: This is required due to the small size of the vena cava in a mouse.</li>
	<li>Use post deconvolution images to generate a blood input function time-activity curve as described above.</li>
</ol>
6. Kinetic Modeling
NOTE: The FDG two-tissue compartment model (Figure 1) requires the plasma input function.
<ol>
	<li>Convert the blood input function in the CSV file to the plasma input function using the following equation10: Input_plasma = Input_blood &#215; (0.386e&#8722;0.191t + 1.165).</li>
	<li>In the kinetic modelling tool click the &#39;Kinetic&#39; button. Import the tissue and plasma total activity count CSV files into the kinetic modelling tool by selecting &#39;Load Time Activity Curve&#39; from the &#39;Menu&#39;.</li>
	<li>In the &#39;Model&#39; menu select 2 tissue compartments. Ensure that the box next to k4 is unselected and enter a value of 0. For initial fitting, uncheck the box for vB (blood volume fraction) and enter a value of 2%.</li>
	<li>Click the &#39;Fit current region&#39;. Correct the extracted region of interest for dispersion11,12. Achieve this by minimizing the Chi-square value for the FDG model for different dispersion times.</li>
	<li>Perform a second fit using floating vB value (check the box next to the box for vB) and the optimized dispersion value to calculate the regional rate constants (k1-k3). Calculate the regional influx constant as Ki = (k1 &#215; k3) / (k2 + k3).</li>
</ol>

<ol>
	<li>Jensen, M. M., Kjaer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+anti-cancer+treatment+with+(18)F-FDG+and+(18)F-FLT+PET:+a+comprehensive+review+of+pre-clinical+studies.">Monitoring of anti-cancer treatment with (18)F-FDG and (18)F-FLT PET: a comprehensive review of pre-clinical studies.</a> Am J Nucl Med Mol Imaging. 5, 431-456 (2015).</li><li>Duncan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(18)F-FDG-PET/CT+imaging+in+an+IL-6-+and+MYC-driven+mouse+model+of+human+multiple+myeloma+affords+objective+evaluation+of+plasma+cell+tumor+progression+and+therapeutic+response+to+the+proteasome+inhibitor+ixazomib.">(18)F-FDG-PET/CT imaging in an IL-6- and MYC-driven mouse model of human multiple myeloma affords objective evaluation of plasma cell tumor progression and therapeutic response to the proteasome inhibitor ixazomib.</a> Blood Cancer J. 3, e165 (2013).</li><li>Wang, Y., Kung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18F-FDG-PET/CT+imaging+of+drug-induced+metabolic+changes+in+genetically+engineered+mouse+lung+cancer+models.">18F-FDG-PET/CT imaging of drug-induced metabolic changes in genetically engineered mouse lung cancer models.</a> Cold Spring Harb Protoc. 2015, 176-179 (2015).</li><li>Wang, X., Minze, L. J., Shi, Z. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+brown+fat+in+mice+with+18F-FDG+micro-PET/CT.">Functional imaging of brown fat in mice with 18F-FDG micro-PET/CT.</a> Journal of visualized experiments : JoVE. , (2012).</li><li>Radu, C. G., Shu, C. J., Shelly, S. M., Phelps, M. E., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positron+emission+tomography+with+computed+tomography+imaging+of+neuroinflammation+in+experimental+autoimmune+encephalomyelitis.">Positron emission tomography with computed tomography imaging of neuroinflammation in experimental autoimmune encephalomyelitis.</a> Proc Natl Acad Sci U S A. 104, 1937-1942 (2007).</li><li>Toba, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-natal+treatment+by+a+blood-brain-barrier+permeable+calpain+inhibitor,+SNJ1945+rescued+defective+function+in+lissencephaly.">Post-natal treatment by a blood-brain-barrier permeable calpain inhibitor, SNJ1945 rescued defective function in lissencephaly.</a> Sci Rep. 3, 1224 (2013).</li><li>Halseth, A. E., Bracy, D. P., Wasserman, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+hexokinase+II+increases+insulinand+exercise-stimulated+muscle+glucose+uptake+in+vivo.">Overexpression of hexokinase II increases insulinand exercise-stimulated muscle glucose uptake in vivo.</a> Am J Physiol. 276, E70-E77 (1999).</li><li>Defronzo, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Banting+Lecture.+From+the+triumvirate+to+the+ominous+octet:+a+new+paradigm+for+the+treatment+of+type+2+diabetes+mellitus.">Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus.</a> Diabetes. 58, 773-795 (2009).</li><li>Tohka, J., Reilhac, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconvolution-based+partial+volume+correction+in+Raclopride-PET+and+Monte+Carlo+comparison+to+MR-based+method.">Deconvolution-based partial volume correction in Raclopride-PET and Monte Carlo comparison to MR-based method.</a> NeuroImage. 39, 1570-1584 (2008).</li><li>Wu, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.+In+vivo+quantitation+of+glucose+metabolism+in+mice+using+small-animal+PET+and+a+microfluidic+device.">et al. In vivo quantitation of glucose metabolism in mice using small-animal PET and a microfluidic device.</a> J Nucl Med. 48, 837-845 (2007).</li><li>Iida, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Error+analysis+of+a+quantitative+cerebral+blood+flow+measurement+using+H2(15)O+autoradiography+and+positron+emission+tomography,+with+respect+to+the+dispersion+of+the+input+function.">Error analysis of a quantitative cerebral blood flow measurement using H2(15)O autoradiography and positron emission tomography, with respect to the dispersion of the input function.</a> J Cereb Blood Flow Metab. 6, 536-545 (1986).</li><li>Cochran, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+PET+imaging+with+[18F]FDG+to+explain+improved+glucose+uptake+in+an+apolipoprotein+A-I+treated+mouse+model+of+diabetes.">In vivo PET imaging with [18F]FDG to explain improved glucose uptake in an apolipoprotein A-I treated mouse model of diabetes.</a> Diabetologia. 59, 1977-1984 (2016).</li><li>Kobayashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+db/db+mouse,+a+model+for+diabetic+dyslipidemia:+molecular+characterization+and+effects+of+Western+diet+feeding.">The db/db mouse, a model for diabetic dyslipidemia: molecular characterization and effects of Western diet feeding.</a> Metabolism. 49, 22-31 (2000).</li><li>Yue, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+progressive+cardiomyopathic+changes+in+the+db/db+mouse.">Magnetic resonance imaging of progressive cardiomyopathic changes in the db/db mouse.</a> Am J Physiol Heart Circ Physiol. 292, H2106-H2118 (2007).</li><li>Hagberg, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+VEGF-B+as+a+novel+treatment+for+insulin+resistance+and+type+2+diabetes.">Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes.</a> Nature. 490, 426-430 (2012).</li><li>Alf, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+brain+glucose+metabolism+by+18F-FDG+PET+with+real-time+arterial+and+image-derived+input+function+in+mice.">Quantification of brain glucose metabolism by 18F-FDG PET with real-time arterial and image-derived input function in mice.</a> J Nucl Med. 54, 132-138 (2013).</li><li>Tantawy, M. N., Peterson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+[18F]FDG+image-derived+input+function+using+the+left+ventricle,+liver,+and+one+venous+blood+sample.">Simplified [18F]FDG image-derived input function using the left ventricle, liver, and one venous blood sample.</a> Molecular imaging. 9, 76-86 (2010).</li><li>Thorn, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeatable+noninvasive+measurement+of+mouse+myocardial+glucose+uptake+with+18F-FDG:+evaluation+of+tracer+kinetics+in+a+type+1+diabetes+model.">Repeatable noninvasive measurement of mouse myocardial glucose uptake with 18F-FDG: evaluation of tracer kinetics in a type 1 diabetes model.</a> J Nucl Med. 54, 1637-1644 (2013).</li><li>Wagner, R., Zimmer, G., Lacko, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+interspecies+approach+to+the+investigation+of+the+red+cell+membrane+glucose+transporter.">An interspecies approach to the investigation of the red cell membrane glucose transporter.</a> Biochim Biophys Acta. 771, 99-102 (1984).</li><li>Flores, J. E., McFarland, L. M., Vanderbilt, A., Ogasawara, A. K., Williams, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+anesthetic+agent+and+carrier+gas+on+blood+glucose+and+tissue+uptake+in+mice+undergoing+dynamic+FDG-PET+imaging:+sevoflurane+and+isoflurane+compared+in+air+and+in+oxygen.">The effects of anesthetic agent and carrier gas on blood glucose and tissue uptake in mice undergoing dynamic FDG-PET imaging: sevoflurane and isoflurane compared in air and in oxygen.</a> Mol Imaging Biol. 10, 192-200 (2008).</li></ol>

The authors have nothing to disclose.

The protocol described here represents a robust, non-invasive methodology to determine the kinetics of glucose uptake from the blood stream into tissue and subsequent metabolism in mice.
The db/db mouse is a is a well-established animal model of type 2 diabetes14 that has been used extensively to examine insulin resistance and relevant interventions. However, previous studies have only quantified endpoint uptake in the heart15 and cardiac and skeletal muscle16.
The use of kinetic analysis to determine physiological rate constants and model the uptake of 18F-FDG from plasma into tissue allows for insights into the impact of antidiabetic treatments on glucose uptake and metabolism. In addition, these experiments can be performed longitudinally to assess, for example, the impact of age or diet on glucose metabolism. This is advantageous over traditional methods that require euthanasia and collection of organs of interest and thus provide information only at a single time point.
Whilst input functions have previously been determined using the whole heart17 as well as heart, liver and a blood samples18 in mice, the protocol described here permits calculation of the input function using a region of interest over the vena cava19. It is also possible to calculate input functions using arterial blood samples during a PET study. However, this is impractical due to the small blood volume of mice.
The use of a plasma input function rather than the 18F-FDGsignal in whole blood is due to the uptake of 18F-FDG into mouse red blood cells20. Furthermore, the red blood cell associated activity represents 18F-FDG inside red cells, and hence is not readily available for transport into other tissue compartments.
In this protocol, it is critical to ensure correct placement of the catheter placed into the tail vein for delivery of the 18F-FDG bolus to the vena cava and throughout the body of the mouse. Warming of the tail to dilate the tail vein significantly improves the ease of inserting this catheter. It is also important to ensure that the PET and CT images are properly coregistered so that the ROIs drawn on the CT image correctly correspond to the PET signal.
There is some debate over the choice of anesthetic agent in studies examining glucose uptake. Whilst isoflurane is a commonly used veterinary anesthetic, the use of sevoflurane may be advantageous in 18F-FDG PET experiments21. In this protocol, it is important to ensure that any potential bias associated with isoflurane anesthesia is minimized by keeping the time between induction of anesthesia and the commencement of imaging constant.

The purpose of this study was to develop a positron emission tomography/computed tomography (PET/CT) based methodology to quantify the in vivo, real-time uptake of glucose from the blood stream into specific tissues in mice. This was achieved using 18F-labelled fluorodeoxyglucose (FDG) to measure glucose uptake and kinetic modelling to estimate the rates of 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.
In rodents, 18F-FDG has been used in the pre-clinical assessment of numerous cancer treatments1, studies of tumor progression2 and tumor metabolism3 as well as imaging of brown fat depots4, neuroinflamation5 and brain metabolism6.
Traditional methods used to examine the tissue specific uptake of glucose in mice (and rats) generally involve treatment with 2-deoxyglucose radiolabeled with either 3H or 14C followed by euthanasia, tissue collection and measurement of radioactivity in each tissue7. The use of PET/CT allows for noninvasive determination of glucose uptake and metabolism in multiple organs and regions simultaneously in live animals. Additionally, as euthanasia is not a requirement, this technique is suitable for use in longitudinal studies.
Type 2 diabetes mellitus (T2DM) is characterized by disrupted glucose metabolism and hyperglycemia secondary to reduced tissue responsiveness to insulin (insulin resistance) and the inability of pancreatic -cells to produce adequate amounts of insulin8. Kinetic analysis of glucose uptake and metabolism can provide important insights into the mechanism of action and efficacy of therapeutic interventions as well as allow for advanced monitoring of disease progression.

<table border="0" cellpadding="0" cellspacing="0" width="654">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PET/CT Scanner</td>
			<td style="width:152px;">Siemens</td>
			<td style="width:119px;">Inveon&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">18F-FDG</td>
			<td style="width:152px;">PETNET Solutions</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td style="width:152px;">Pharmachem</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30 guage needle</td>
			<td style="width:152px;">BD</td>
			<td align="right" style="width:119px;">305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PMOD modelling software</td>
			<td style="width:152px;">PMOD Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BKS.Cg-Dock7m&#160;+/+&#160;Leprdb/J&#160; mice</td>
			<td style="width:152px;">Jackson Laboratory</td>
			<td style="width:119px;">000642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Human insulin</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We have previously used the db/db mouse model to investigate the impact of increasing plasma apoA-I levels on the kinetics of glucose uptake and metabolism13. In this study we used db/db mice treated with insulin to demonstrate the utility of PET/CT imaging to monitor the uptake of 18F-FDG from the plasma into the gastrocnemius muscle in real time.
Six week old db/db mice were anesthetized and treated with 3 U human insulin/kg, or equivalent volume PBS via subcutaneous injection 30 min before the start of the PET scan. Mice received 10 MBq of 18F-FDG via intravenous injection and uptake measured by PET for 60 min. A CT scan was performed for anatomical reference.
Regions of interest were drawn on the vena cava and gastrocnemius muscle using the CT images (Figure 2). Treatment of db/db mice with insulin increased the 18F-FDG activity in the gastrocnemius ROI over the acquisition time period (Figure 3A). The values obtained for the vena cava ROI were converted from blood to plasma values and were not altered by insulin treatment in insulin treated mice relative to control (Figure 3B).
Time activity curves were loaded into the kinetic modelling tool for calculation of kinetic parameters. Data was initially fitted to a two-tissue compartment method with k4=0 with a Vb (blood volume fraction) value of 2% to calculate dispersion values (80 s and 70 s for PBS and insulin treated mice, respectively). Fitting was then performed using the above dispersion values and a floating Vb value.
No significant difference in the rate of 18F-FDG transport from the arterial plasma to the intracellular space (k1) or from the intracellular space to plasma (k2) was observed in insulin treated mice compared to control (Table 1). The rate of 18F-FDG phosphorylation (k3) was significantly increased in insulin treated mice (7.06 &#177; 6.60 &#215; 10-3 vs 2.26 &#177; 0.72 &#215; 10-2 min-1 for PBS and insulin treated groups, respectively; p &#60; 0.05). Insulin treatment also significantly increased the influx constant (Ki) compared to the PBS-treated animals (5.51 &#177; 4.25 &#215; 10-4 vs 2.01 &#177; 0.28 &#215; 10-3 mL min-1 g-1, respectively; p &#60; 0.05).
<img alt="Figure 1" src="/files/ftp_upload/55184/55184fig1.jpg" /> 
Figure 1: Regional time-activity curves were fitted to a two-tissue, three compartment model, with C1 being the concentration of FDG in plasma and C2 and C3 the concentration of FDG and phosphorylated FDG in tissue, respectively. k1 represents the FDG uptake rate in tissue, k2 the clearance rate from the tissue back to the plasma compartment and k3 the phosphorylation rate of FDG. The dephosphorylation of FDG was assumed to be negligible (k4=0).
<img alt="Figure 2" src="/files/ftp_upload/55184/55184fig2.jpg" /> 
Figure 2: Example of drawing of regions of interest in gastrocnemius muscle (A) and vena cava (B) on a co-registered PET-CT image. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55184/55184fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55184/55184fig3.jpg" /> 
Figure 3: Time-activity curves for regions of interest in gastrocnemius muscle (A) and vena cava (B). Male db/db mice were fasted for 4.5 h prior to receiving 3 U/kg insulin (red) or equivalent volume PBS (black). 18F-FDG (10 MBq) was delivered via intravenous injection and 18F-FDG levels in the gastrocnemius muscle and vena cava determined by PET/CT for 60 min. Values are mean &#177; SD and expressed as percentage of injected dose, calculated from a full field of view (n=4/group). Modified from Cochran et al. 13 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55184/55184fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Treatment</td>
			<td>k1</td>
			<td>k2</td>
			<td>k3</td>
			<td>Ki</td>
		</tr>
		<tr>
			<td>(mL min-1 g-1)</td>
			<td>(min-1)</td>
			<td>(min-1)</td>
			<td>(mL min-1 g-1)</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>1.31&#177;0.42&#215;10-2</td>
			<td>0.12&#177;0.11</td>
			<td>7.06&#177;6.60&#215;10-3</td>
			<td>5.51&#177;4.25&#215;10-4</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>1.45&#177;0.59&#215;10-2</td>
			<td>0.09&#177;0.05</td>
			<td>2.26&#177;0.72&#215;10-2 *</td>
			<td>2.01&#177;0.28&#215;10-3 *</td>
		</tr>
	</tbody>
</table>
Table 1: Kinetic analysis of increased 18F-FDG from plasma to gastrocnemius muscle in insulin treated db/db mice. Values are mean &#177; SD. *p&#60;0.05 vs PBS according to a Mann-Whitney test (n=4/group).

Watch this Scientific Journal Video about Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT at JoVE.com

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.

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). ...

determining-glucose-metabolism-kinetics-using-18f-fdg-micro-petct

Nanyang Technological University

Research

JoVE Journal

Medicine

13.8K Views.  UNSW Australia. 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.

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

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

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 ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

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 ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of disorders of consciousness, disrupted higher brain function, and verbal disturbances. As a consequence of an insufficient ability to communicate, objective evaluations are needed from family members, medical staff, and caregivers. One such evaluation is the assessment of functioning brain areas. Recently, multimodal brain imaging has been used to explore the function of damaged brain areas. [18F]-fluorodeoxyglucose positron emission tomography-computed tomography ([18F]FDG-PET/CT) is a successful tool for examining brain function. However, the assessment of brain glucose metabolism based on [18F]FDG-PET/CT is not standardized and depends on several varying parameters, as well as the patient&#39;s condition. Here, we describe a series of semiquantitative assessment protocols for a region-of-interest (ROI) image analysis using self-produced [18F]FDG tracers in patients with sTBI. The protocol focuses on screening the participants, preparing the [18F]FDG tracer in the hot lab, scheduling the acquisition of [18F]FDG-PET/CT brain images, and measuring glucose metabolism using the ROI analysis from a targeted brain area.

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

[18F]-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography is useful for studying glucose metabolism related to brain function. Here, we present a protocol for an [18F]FDG tracer set-up and semiquantitative assessment of the region-of-interest analysis for targeted brain areas associated with clinical manifestations in patients with severe traumatic brain injury.

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...