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Basic Science Investigation |
The Division of Radiological Sciences, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri
Correspondence: For correspondence contact: Pilar Herrero, MS, Mallinckrodt Institute of Radiology, Washington University School of Medicine, Campus Box 8225, 510 S. Kingshighway Blvd., St. Louis, MO 63110. E-mail: herrerop{at}mir.wustl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Key Words: metabolism • diabetes mellitus • fatty acids • imaging • PET
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Results of studies in small animals are now showing that alterations in myocardial substrate metabolism plays a key role in variety of normal and abnormal cardiac states, such as normal aging, hypertension-induced left ventricular hypertrophy, obesity, and diabetic heart disease. For example, we have previously shown that it is possible to qualitatively show differences in cardiac metabolism in a transgenic model of diabetes in which PPAR
(peroxisome proliferator-activated receptor ) is upregulated (2). Thus, the ability to quantify myocardial metabolism in both normal and diseased rodents will have direct ramifications on the development of potential therapies and their translation into clinical practice (3).
The advent of small-animal PET technology allows for the sequential measurements of cardiac metabolic rates in the same animal (4–6). This current feasibility study was aimed at validating the use of PET to address the general hypothesis that rat models of cardiac disease in conjunction with small-animal PET can be used to quantify alterations in myocardial substrate metabolism in the rat myocardium. In this study we used a rat model of type 2 diabetes mellitus (Zucker diabetic fatty [ZDF] rats) to examine the feasibility of using small-animal PET, and kinetic modeling techniques extensively validated in large animals (7–11) and currently used in humans (12–16), to detect noninvasively changes in myocardial substrate metabolism in vivo in rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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-counter. Small-animal PET was performed on either the microPET R4 (20) or the microPET Focus (21) tomograph (Concorde Microsystems Inc.) housed in a temperature-controlled imaging suite.
Animal Preparation
All animal experiments were conducted in compliance with the Guidelines for the Care and Use of Research Animals established by Washington University's Animal Studies Committee. Optimal animal handling methods for small-animal PET have been extensively studied by our group and have been reported elsewhere (22). Ten male ZDF rats (fa/fa) and their corresponding matched lean littermates (Lean, Fa/+), from Charles River Laboratories, Inc. (Table 1), were fed Purina Constant Nutrition 5008, consisting of 26.8% protein, 16.7% fat, and 56.4% carbohydrates. Before the imaging studies, rats were placed in metabolism cages and were fasted from food for 6 h with water being given ad lib. On each study day the rats were anesthetized with 2%–2.5% isoflurane inhaled anesthesia via an induction chamber. Maintenance of anesthesia throughout the procedure consisted of 1%–1.5% isoflurane via a custom-designed nose cone. The rat's neck was shaved and scrubbed, in preparation for a sterile cutdown procedure. A 1- to 1.5-cm incision was made over the right jugular vein. The vein was exposed and ligated, and a microrenathane catheter (0.025-mm outer diameter x 0.012-mm inner diameter) was sutured in place. The rat was globally heparinized (10 mg/kg) to prevent the catheter from clotting. Body temperature was maintained using a circulating water blanket as well as a heat lamp. Pediatric electrocardiogram leads (Red Dot Infant Electrodes; M.M.M. Co.) were placed on the rat's hind limbs to measure and record heart rate.
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Metabolite Measurements
During each metabolic imaging session, arterial whole blood samples were taken at 2, 5, 10, 15, and 20 min after each tracer injection to measure the contribution of key 11C-metabolites to the total blood radioactive counts (11CO2, %) for 1-11C-acetate and 1-11C-palmitate and total acidic metabolites (TAM, %) for 1-11C-glucose). TAM (11CO2 and 11C-lactate) were separated from neutral and basic species using resin columns as previously reported (22). The total 11C-blood activity in each blood sample was measured before being placed on the resin column and was used in conjunction with factor analysis (FA) blood input function measurements.
Kinetic Modeling
Myocardial perfusion and metabolism were measured using compartmental models describing the kinetics of 15O-water (9,23), 1-11C-glucose (7,11), 1-11C-acetate (10), and 1-11C-palmitate (8). These models have been extensively validated in large-animal studies and are currently being used in our institution to assess intermediary metabolism in clinical research studies (12–16). The validity of using 15O-water for determining myocardial blood flow (MBF) in the rat heart has been shown by comparison with radioactive microspheres (24).
To define the kinetics of each tracer, blood (input function) and myocardial tracer activity over time (time–activity curve, counts/pixel/min) were extracted from myocardial sequential PET images using FA methods (25,26), which have been previously validated for use in rats (27). For a given study, the same 4 or 5 blood and myocardial midventricular transaxial slices were selected manually for each tracer and added to create a 3-dimensional (3D) volume to obtain 3 "pure" factors: right ventricle, left ventricle and myocardium. Because the animals remained under anesthesia for the entire GAP study, it was assumed that the 3D volume generated encompassed the same anatomic volume in all 4 tracers. Two factors, the left ventricular (input function) and myocardial time–activity curves, were then used in conjunction with the appropriate compartmental kinetic models to obtain measurements of perfusion, overall oxidation, and intermediary substrate metabolism for each animal. Because only one global myocardial time–activity curve was generated per tracer, only global—not regional—measurements were obtained.
The number of compartments that are needed to quantify the fate of the tracer in myocardium is directly proportional to the number of steps in the metabolic process being investigated. Compartments are usually represented by boxes or circles and are linked together by arrows representing possible pathways the tracer can follow in a particular metabolic pathway (Fig. 3). Each compartment in a kinetic model is defined as the space in which the tracer is uniformly distributed. The symbol k above or beside each arrow in Figure 3 represents tracer transfer rate constant and denotes the fraction of the total tracer that leaves the compartment per unit time (min–1). The 2 key assumptions of compartmental modeling—tracer is homogeneously distributed in each compartment and the rate of tracer transfer from compartment 1 to compartment 2 is directly proportional to the tracer concentration in compartment 1—make it possible to describe the system under study by n first-order, linear differential equations. Each differential equation represents the rate of change of tracer concentration over time in compartment n. The solution to the system of differential equations gives the concentration of tracer in each compartment over time (qn, g/mL). The sum of the concentrations from all compartments (a function of the tracer's blood activity and the turnover rates) represents the myocardial activity over the scanning period (theoretic myocardial time–activity curve). Using numeric nonlinear least-square approaches, turnover rates (k values) can then be estimated by minimizing the differences between the theoretic myocardial time–activity curve predicted by the model and the myocardial time–activity curve obtained from FA of PET images.
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Oxygen consumption (MVO2, µmol/g/min) was estimated from the modeling transfer rate representing 1-11C-acetate oxidation (clearance rate). For the metabolic studies (1-11C-glucose and 1-11C-palmitate), the estimated transfer rates obtained from kinetic modeling were first used to calculate myocardial extraction of tracer (for both 1-11C-glucose [MGEF] and 1-11C-palmitate [MFAEF]) and fractional oxidation [EF-O] and esterification [EF-S] (for 1-11C-palmitate). Myocardial glucose uptake and utilization (MGUP, MGU) and myocardial fatty acid uptake and utilization (MFAUP, MFAU), as well as myocardial fatty acid oxidation (MFAO) and esterification (MFAE) (16), respectively, were calculated as follows:
 | (Eq. 1) |
 | (Eq. 2) |
Similarly, MFAO and MFAE were also calculated from the kinetics of 1-11C-palmitate as follows:
 | (Eq. 3) |
 | (Eq. 4) |
One of the unique features of the noninvasive PET methodology used to assess myocardial substrate (FFA or glucose) utilization lays in its ability to differentiate peripheral from myocardial changes in cardiac substrate utilization. Measurements of total myocardial substrate utilization (nmol/g/min) (Eq. 2), as measured by PET, is the product of myocardial substrate uptake (mL/g/min) (derived from kinetic modeling, Eq. 1) and blood substrate levels (measured from blood samples). Thus, changes in substrate utilization can be due to substrate changes in blood (peripheral effect) or changes in myocardial substrate uptake (myocardial effect).
Statistical Analysis
Mean differences in perfusion and metabolic measurements between ZDF rats and their lean controls were determined by the Student t test. P values < 0.05 were considered statistically significant. All group values are presented as mean ± SD. The coefficient of variation during the GAP study (COV, %) for hemoglobin A1c (HbA1c), insulin, glucose, FFA, respiration rate, and heart rate was defined as 100·SD/mean.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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2-fold elevation in plasma insulin, glucose, and FFA levels compared with lean animals. The variability of plasma insulin and glucose (COV in Table 1) during the study period were comparable for both groups. In contrast, the FFA COV was significantly higher in the ZDF group (COV in Table 1). Although there were no differences in heart rate between ZDF and lean animals and MBF, the respiration rate (RR) was significantly higher and the MVO2 tended to be significantly lower in the ZDF group. The high RR observed in the ZDF rats was most probably due their susceptibility to acidosis resulting from the reinhalation of expelled CO2 from the cone piece placed in the animal's snout during the study. An example of microPET images obtained from the 3 metabolic tracers is shown in Figure 2. The quality of the images was similar between the hearts of the lean and the ZDF rats. FMM and FBM values estimated from 15O-water kinetics were not significantly different between ZDF and Lean (FMM-w, 0.76 ± 0.20 vs. 0.72 ± 0.19, and FBM-w, 0.27 ± 0.14 vs. 0.27 ± 0.15) rats. There were no differences in FBM values between ZDF and lean rats obtained from 1-11C-acetate kinetics (FBM-ac, 0.17 ± 0.11 vs. 0.12 ± 0.11; P = not significant [NS]), 1-11C-palmitate kinetics (FBM-pal, 0.29 ± 0.24 vs. 0.20 ± 0.16; P = NS), or 1-11C-glucose kinetics (FBM-glu, 0.30 ± 0.28 vs. 0.31 ± 0.23; P = NS).
The results obtained in the lean and ZDF rats for myocardial glucose and FFA metabolism are shown in Figures 4 and 5, respectively. In spite of the ZDF group having 2-fold higher levels of insulin than their controls (Table 1), MGUP was not significantly different between the 2 groups (Lean, 0.044 ± 0.041, vs. ZDF, 0.068 ± 0.051 mL/g/min; P = NS). However, because of high levels of plasma glucose in the ZDF group (Lean, 10.7 ± 4.4, vs. ZDF, 28.1 ± 4.9 µmol/mL; P < 0.0001) (Fig. 4), MGU tended to be higher in the ZDF group (Lean, 629 ± 785, vs. ZDF, 1,737 ± 1,406 nmol/g/min; P = 0.06). During the 1-11C-palmitate study, though MFAUP was not significantly different between the 2 groups (Lean, 0.51 ± 0.28, vs. ZDF, 0.72 ± 0.19 mL/g/min), plasma FFA levels were significantly higher in the ZDF animals when compared with their lean littermates (Lean, 1,052 ± 497, vs. ZDF, 2,277 ± 789 nmol/mL; P < 0.0005), resulting in a 3-fold higher MFAU in the ZDF group (Lean, 520 ± 462, vs. ZDF, 1,623 ± 712 nmol/g/min; P < 0.001). Consistent with higher MFAU, the ZDF rats showed higher levels of MFAO when compared with their controls (Lean, 453 ± 478, vs. ZDF, 1,363 ± 730 nmol/g/min; P < 0.01). No significant differences were observed between groups in the fraction of MFAU that was oxidized most probably due to the large variability of the measurement observed in the lean group (% of MFAO/MFAU: Lean, 79 ± 29, vs. ZDF, 91 ± 4; P = NS) (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Because of the small size of the rat heart we were unable to perform conventional arteriovenous balance studies that are typically used to verify the accuracy of new PET radiotracer techniques in studies of large animals or humans (8,11,30). Acute tissue autoradiographic methods could have been used to evaluate the absolute retention of radiotracer in rat heart. However, this method does not provide the necessary temporal information to permit the comparison with our PET kinetic models. Consequently, we performed this proof-of-concept study by using the ZDF rat model, which faithfully recapitulates many aspects of the human metabolic syndrome (31). ZDF rats have been used extensively as a model of type 2 diabetes mellitus, examining the multiple aspects of this disease, including prevention, progression, and complications (32). Not only do the majority of young male ZDF rats develop symptoms of the diabetic syndrome—by becoming insulin resistant and later overtly obese—but also they respond well to drugs (e.g., glitazones or metformin) targeted toward the treatment of humans with type 2 diabetes mellitus. ZDF rats also develop a cardiomyopathic phenotype associated with type 2 diabetes mellitus (33). Although the cause(s) of this phenotype is unclear, the myopathy is hypothesized to be at least partially the result of the increase in myocardial fatty acid metabolism (33). With the advent of noninvasive small-animal PET technologies and the validated use of GAP PET studies to delineate myocardial metabolism in humans, we undertook the current feasibility study to establish the practicality and efficacy of imaging quantitative changes in the myocardial metabolism of ZDF rats as the diabetic syndrome develops.
The ZDF animals were studied within 2 mo from the onset of diabetes; thus, observations of metabolic changes in these animals should be consistent with the metabolic changes that take place during the early stages of type 2 diabetes mellitus. The lean littermates acted as controls against the ZDF animals and demonstrated the anticipated alterations in HbA1c, insulin, glucose, and FFA levels consistent with the advent of type 2 diabetes mellitus in the ZDF rats at the time of study. The results of this feasibility study are consistent with the well-known metabolic changes that take place in the heart of the ZDF rat. Simply stated, when compared with their lean controls, the ZDF rat showed (a) elevated insulin, glucose, and FFA levels; (b) elevated FFA utilization and oxidation; (c) preserved myocardial glucose utilization; and (d) a trend toward a lower MVO2. The metabolic data obtained noninvasively in this study using small-animal PET and well-established kinetic modeling approaches is consistent with the well-known early metabolic derangements of the diabetic heart, which include high fatty acid uptake, utilization, and oxidation that precedes glucose metabolism changes (32,34). For example, Aasum et al. (34), using invasive ex vivo procedures, demonstrated in db/db mice that diabetic hearts exhibit increased fatty acid oxidation that preceded reductions in carbohydrate oxidation. Wang et al. (35) have shown that the rate·pressure product and coronary flow (mL/g/min) were reduced in 12-wk-old isolated hearts of (not paced) male ZDF rats when compared with their lean controls. Their animals were fed the same chow as in this study (Purina 5008), were similar in age and weight, and were studied under glucose and insulin plasma levels comparable with ours. In a subsequent study in which isolated hearts of 12-wk-old (paced) ZDF rats and their lean controls were studied under conditions to mimic the fed state, Wang et al. (36) observed that, at rest, the ZDF rats exhibited higher FFA oxidation and lower lactate, and, to a lesser extent, pyruvate oxidation, with no differences in glucose oxidation and MVO2. Furthermore, they observed no differences in oxidative ATP production between ZDF and control rats.
Results of studies in isolated perfused hearts (37) as well as in vivo (38) have shown that the glucose transporter GLUT-4 is downregulated in 8-wk-old (37) and 12-wk-old (38) ZDF rats, resulting in lower glucose uptake (38) and oxidation (37) in the ZDF rats when compared with their lean controls. In the present study, when compared with their lean controls, we observed comparable levels of glucose uptake in the ZDF rats in spite of 2-fold higher insulin levels in these animals. Moreover, high glucose blood levels resulted in a strong trend toward higher glucose utilization in the ZDF rats. However, measurements of myocardial substrate metabolism were performed under fasting conditions, which result in FFA being the primary source of cardiac energy. Under these conditions, myocardial glucose extraction and oxidation by the heart is very low. The low extraction of tracer results in myocardial time–activity curves with low counts and high noise and, consequently, potential poor estimates of myocardial glucose extraction. In the present study, in contrast to myocardial uptake of 1-11C-palmitate, which ranged from 0.03 to 0.95 mL/g/min, myocardial uptake of 1-11C-glucose ranged from <0.01 to 0.13 mL/g/min. The low extraction of 1-11C-glucose by myocardium most likely contributed to the large group variability observed in glucose uptake in both Lean (93% COV) and ZDF (75% COV) rats (Fig. 3). This variability was much larger than the variability observed in glucose uptake measurements done in lean subjects studied under fed conditions (n = 11) and type 1 diabetics studied during an insulin drip (n = 11) using the same modeling methodology (lean, 40% COV; type 1 diabetics, 59% COV) (12). To overcome this problem, studies designed to assess glucose metabolism could be done under conditions of high myocardial glucose use, such as fed or hyperinsulinemic–euglycemic clamping conditions.
The results of this study are consistent with the well-established early onset of cardiac substrate metabolic derangement in the ZDF rat, demonstrating that noninvasive measurements of cardiac substrate metabolism is feasible in rats using kinetic modeling in conjunction with PET tracers and dynamic microPET imaging. However, there are several issues pertaining to the methodology used that need to be addressed. The use of isoflurane for anesthesia in rodents is the preferred method because of the rapid induction and recovery from anesthesia (22). Other anesthetics prevent the possibility of long imaging studies (4 h) and recovery from surgery. Evidence in dogs suggests that, after 10 min of isoflurane, both plasma FFA concentration and palmitate turnover decreased by >70% throughout the remainder of the anesthesia (39). In the same study, glucose production increased after induction of anesthesia and peripheral glucose utilization decreased after 3.5 h of isoflurane anesthesia. These data, therefore, suggest a widespread and immediate metabolic effect during isoflurane anesthesia. However, careful attention to detail, including the monitoring of FFA levels and data based on comparisons between ZDF and lean littermates, as well as the application of accurately defined kinetic models, takes these factors into account.
All kinetic models are implemented under the assumption that the system under study is in steady state. In the present study, although the variability of FFA during microPET imaging was higher in the ZDF group, heart rate, plasma insulin, and glucose variability during microPET imaging were comparable between the ZDF rats and their controls. However, insulin, glucose, and FFA variability was relatively large (Table 1), partially because of the length of the imaging studies (4–6 h). To minimize these relatively large physiologic variations in insulin and substrate levels, interventions such as euglycemic–hyperinsulinemic clamp or constant infusion of low-dose intralipid might be desirable.
The poor resolution of current microPET scanners (
2.0-mm full width at half maximum) in relation to the very small dimensions of the rat heart (approximately 2-mm wall thickness and 5.5-mm left ventricular diameter) results in severe distortion of blood and myocardial time–activity curves generated from dynamic cardiac microPET images. Blood (input function) and myocardial time–activity curves—the key data required for kinetic modeling—must be corrected for these resolution effects. In the present study both blood and myocardial time–activity curves for all tracers used were extracted from microPET cardiac images by FA. This approach results in uncontaminated blood time–activity curves and myocardial time–activity curves with relatively low contamination (FMM-w [n = 10] = 0.90 ± 14; FBM-all [n = 40] = 0.11 ± 11). However, this approach does not allow for the extraction of regional myocardial curves, because only 1 global blood and 1 global myocardial curve per tracer are generated. Our group is currently implementing new reconstruction algorithms as well as cardiac gating strategies designed to minimize these resolution effects so that regional analysis of data could be implemented.
Although the results of this feasibility study are consistent with what is known about the myocardial changes in substrate metabolism that occur in the early onset of diabetes, the relatively large variability among animals in substrate and insulin levels (Table 1) as well as myocardial substrate metabolism (Figs. 4 and 5) observed in both lean and ZDF rats is multifactorial. These factors include natural physiologic variability, fluctuations in glucose and FFA metabolism associated with isoflurane anesthesia as well as with the long duration of the studies (4–6 h), and the uncertainties in measurements associated with the complex modeling approaches implemented, such as noise of PET data, estimation (vs. direct measurement) of model parameters and partial-volume and spillover effects, and correction of blood PET data for blood 11C-metabolites. Thus, to optimize these complex studies, by removing or minimizing some of these sources of error, more systematic studies are required. These studies should include sequential studies in the same animal in which substrate and insulin levels are maintained constant during PET data acquisition and the use of different anesthetics is investigated. Once the animal protocol is optimized, uncertainty and variability of the measurements obtained from these modeling approaches could be further evaluated and optimized by continuing to develop methods to improve data obtained from a small-animal PET scanner (by minimizing partial-volume and spillover effects and enhancing signal to noise ratios), such as the implementation of respiratory gating and new reconstruction algorithms.
It is also important to note that the studies presented here have been performed in a rat model of cardiac disease, and translation to the mouse (the most common form of transgenic model) will require an additional stage of validation. The smaller size of the mouse myocardium will exacerbate partial-volume and spillover effects. Spatial resolution and sensitivity may be issues if syndromes such as ischemia are to be examined. We are continuing to develop methods to use with the mouse myocardium, such as the implementation of respiratory gating and new reconstruction algorithms to overcome these potential limitations.
Implementation of the current methodology is challenging as it requires a wide range of expertise and resources, such as animal technologists, radiochemists, physicists, mathematical modelers, and data analysts as well as access to a cyclotron and the ability to produce on demand the radiotracers used. In spite of these challenges, our institution is currently implementing these methods successfully in clinical studies designed to elucidate the potential impact that alterations in cardiac substrate metabolism attributed to different diseases, such as diabetes and cardiomyopathy, have on cardiac function. Nonetheless, the quantification of myocardial perfusion and metabolism in small animals is not limited to the use of the tracers used in the present study. Following the work presented in this article, other tracers to quantify metabolism and other biologic processes in the myocardium and other tissues can be validated in a similar manner.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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ACKNOWLEDGMENTS |
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References |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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overexpression mimics that caused by diabetes mellitus. J Clin Invest. 2002;109:121–130.[Medline]Related articles in JNM:
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, ZDF; 
, Lean).