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AJR 2005; 184:658-662
© American Roentgen Ray Society

MRI of Fat Distribution in a Mouse Model of Lysosomal Acid Lipase Deficiency

Hong Du1,2, Bernard J. Dardzinski1,2,3, Kendall J. O'Brien3 and Lane F. Donnelly1,2,3

1 Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229-3039.
2 University of Cincinnati College of Medicine, Cincinnati, OH.
3 Department of Radiology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039.

Received March 31, 2004; accepted after revision June 1, 2004.

 
Address correspondence to L. F. Donnelly.


Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
OBJECTIVE. We assessed the use of MRI in the evaluation of abdominal fat distribution in a lysosomal acid lipase (LAL)–deficient mouse model.

MATERIALS AND METHODS. LAL-deficient mice are born with a normal fat distribution but over time deplete the fat stores in the subcutaneous and retroperitoneal tissues and accumulate fat in the liver, spleen, and bowel. Four MRI studies of LAL-deficient mice and control mice were obtained with 3-T T1-weighted spin-echo images and volume segmentation processing to create parameters for the study of fat distribution: intraabdominal adipose tissue–subcutaneous adipose tissue (IAT/SAT) ratio, liver volume, reproductive fat, and retroperitoneal fat. MRI adiposity parameters in LAL-deficient mice were compared with those in control mice. Adiposity volumes calculated on MRI were compared with those calculated at autopsy.

RESULTS. Statistically significant differences were found between LAL-deficient and control mice for IAT/SAT ratio (p = 0.0336), liver volume (p = 0.0336), and reproductive fat (p = 0.0336), and a statistically significant trend was found for retroperitoneal fat (p = 0.0514). No statistically significant difference was found between adiposity volumes calculated on MRI and adiposity volumes found at autopsy (all p > 0.2).

CONCLUSION. Use of an in vivo model showed MRI techniques to be accurate in predicting visceral adiposity. LAL-deficient mice provided a unique model showing a pattern of adipose distribution that is markedly different from that in control mice, and MRI may provide a means of evaluating therapeutic interventions sequentially.


Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The prevalence of obesity in children and adolescents has tripled over the last three decades and, at current estimates of between 15.5% and 37%, is at epidemic proportions in the United States [1, 2].

The distribution of adipose tissue within the abdomen relates strongly to the risk for cardiovascular disease and type 2 diabetes mellitus [3–10]. Individuals with higher volumes of adipose tissue within the viscera (intraabdominal adipose tissue, or IAT), as opposed to within subcutaneous tissues surrounding the abdomen (subcutaneous adipose tissue, or SAT), are at higher risk than patients who store fat primarily in the SAT [3–10]. Measurements of the IAT/SAT ratio have been used to estimate risk factors for cardiovascular disease and type 2 diabetes mellitus, particularly in the setting of research. Currently, the most accurate way to predict the volume of IAT and SAT and calculate the IAT/SAT ratio is with cross-sectional imaging studies, most commonly MRI [7–14].

Multiple studies [3–5] have compared the MRI parameters of visceral adiposity with other risk factors for cardiovascular disease and type 2 diabetes mellitus. One problem with evaluating the accuracy of MRI techniques for visceral adiposity is the lack of a gold standard. No method other than cross-sectional imaging is available to calculate the volume of adipose tissue within the abdominal cavity or subcutaneous tissues for comparison with MRI results in living subjects. A study has evaluated MRI techniques using an in vitro phantom [15]. An in vivo model would be helpful in further evaluating the accuracy of MRI techniques in the prediction of visceral adiposity.

We have access to lysosomal acid lipase (LAL)–deficient mice that can serve as an in vivo model for studying visceral adiposity [16, 17]. The LAL-deficient mice are born with a normal distribution of fat but, soon after birth, begin to deplete normal stores of adipose tissue and accumulate abnormal stores of adipose tissue in multiple organs including the liver, spleen, small intestine, and adrenal glands. This lipodystrophy phenotype provides a model system to monitor adipose tissue changes in vivo using MRI.

We evaluated the use of MRI for monitoring and comparing the distribution of abdominal fat in LAL-deficient and control mice. Our purpose was twofold. The first purpose was to evaluate the use of the LAL-deficient model as an in vivo gold standard for assessing the accuracy of MRI techniques in evaluating visceral adiposity. The second purpose was to use MRI to evaluate differences in fat distribution between LAL-deficient and control mice.


Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The care and treatment of mice were according to institutional guidelines, and all procedures were according to protocols approved by the institutional animal care and use committees. LAL-deficient mice were generated by gene-targeted disruption in embryonic stem cells and originated from mixed genetic backgrounds of 129Sv, CF-1, and FVB/N (25%, 25%, and 50%, respectively) [16]. Genetic background–matched wild-type mice were used as controls. Mice were housed in microisolation, under cycles of 12 hr of light and 12 hr of dark. Water and food (regular chow diet) were available ad libitum. The mice were genotyped by polymerase chain reaction–based screening of tail DNA [16]. Four MRI studies were obtained for each LAL-deficient and control mouse. One control mouse and one LAL-deficient mouse were imaged twice, once at 15 weeks old and once at 30 weeks old. Therefore, the eight MRI examinations were performed on six mice (three LAL-deficient and three control).

Imaging
The mice were imaged on a BioSpec (Bruker) 3-T MRI system using a custom-sized send–receive coil. Coronal spin-echo T1-weighted sequences were obtained through the entire mouse (head to anus). The parameters of the coronal T1-weighted spin-echo images were TR/TE, 500/8; matrix size, 256 x 160; slice thickness, 2 mm; and number of excitations, 2.

The data set was transferred to an in-house computer program for processing. Using the T1-weighted spin-echo data sets, the total adipose tissue (TAT), IAT, SAT, retroperitoneal adipose tissue, and reproductive adipose tissue were calculated [15]. The segmentation software isolates the individual volumes of fat using a k-means clustering algorithm.

The coronal T1-weighted images were segmented into three intensities, with the volume of highest intensity corresponding to the fat region of interest. The volume of high intensity from the limbs and head was excluded from the fat region of interest. The remaining high-intensity volume from the trunk was the TAT. From the TAT, the volume outside the abdominal cavity was excluded and the remaining volume was the IAT. The SAT was calculated by subtracting the IAT from the TAT. The IAT/SAT ratio was then calculated.

The IAT was further subdivided to determine the reproductive and retroperitoneal adipose tissue. Using the IAT, the volume corresponding to the intestine was excluded. From the remaining volume, the retroperitoneal and reproductive fat was identified as a contiguous volume of high intensity corresponding to the location of reproductive and retroperitoneal fat referenced to pictures of wild-type mice with abdomen and back opened to show the reproductive and retroperitoneal fat pads.

Because hepatomegaly develops in LAL-deficient mice, liver volume was calculated also from the coronal T1-weighted images. The liver volume was measured by placing regions of interest around the liver on each individual image that showed liver. For each individual image, the area of the liver was calculated. The thickness of each area was used to calculate the volume of that slice. The individual volumes were then added to calculate the entire liver volume.

Necropsy and Adipose Volume Measurements
Necropsy data were available on five mice (two LAL-deficient and three control; one mouse died before sacrifice). The mice were sacrificed by intraperitoneal injection (0.2 mL) of triple sedative (ketamine, xylazine, and acepromazine), and the adipose tissue for the IAT, retroperitoneal fat, and reproductive fat was dissected and isolated. The adipose tissue for each anatomic region was then weighed (in grams). To calculate the volume of adipose tissue, we divided the weight by the density (specific gravity) of the adipose tissue. The specific gravity was calculated by determining the weight and volume replacement (in phosphate-buffered saline) for a sample of tissue. For the LAL-deficient mice, the density of adipose tissue was 1.2775 g/mL. For the control mice, the density of adipose tissue was 0.8453 g/mL. The difference between the density of adipose tissue from wild-type and LAL-deficient mice reflected the different histology we observed—that is, greater heterogeneity of adipocytes in the adipose tissue of LAL-deficient mice because of the greater ratio of cell membrane mass to lipids [17].

The volume of the liver was also calculated. The density of liver is 0.9774 g/mL for LAL-deficient mice and 0.6983 g/mL for control mice. The difference is most likely related to the different liver composition of the LAL-deficient mice, with 42 times more cholesterol esters than in control mice [17].

Statistical Analysis
Paired t tests were used to determine whether the volumes measured on MRI and at autopsy were statistically different for various anatomic regions. Because even the log transformations of the liver autopsy volume were not normally distributed, a signed rank test was used to analyze liver volume.

With Statistical Analysis Software (SAS Institute), Wilcoxon's signed rank tests were used to evaluate for statistically significant differences in the following MRI parameters between LAL-deficient and control mice: IAT/SAT ratio, liver volume, reproductive fat, and retroperitoneal fat. Statistical Analysis Software provides t or normal approximations. Statistical significance was defined as a p value of less than 0.05.


Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Table 1 compares the volumes of adipose tissue calculated on MRI with the volumes found at autopsy (Fig. 1). No statistically significant differences were found.


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  		TABLE 1 Comparison of Volume of Adipose Tissue Calculated for Various Anatomic Regions from MRI and Autopsy Data

 


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  		Fig. 1. —Photograph of control (left) and lysosomal acid lipase–deficient mouse (right), both approximately 30 weeks old. Depleted subcutaneous adipose tissue, depleted omentum, and markedly enlarged and yellow liver (large arrows) are seen in deficient mouse; normal liver (small arrows) and normal omentum (arrowheads) are seen in control mouse.

 

Table 2 compares the MRI adipose volume data of LAL-deficient mice with the data of control mice. Statistically significant differences were found in IAT/SAT ratio, liver volume, and reproductive fat. These differences are illustrated in Figures 2A, 2B, 3A, 3B, 4A, 4B.


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  		TABLE 2 Comparison of Adipose Volume Parameters Calculated by Volume Segmentation of TI-Weighted MRI Data Between LAL-Deficient and Control Mice

 


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  		Fig. 2A. —Coronal T1-weighted spin-echo MR images obtained with volume segmentation for total adipose tissue. Contiguous images of 30-week-old control mouse show red regions of interest that denote area of total adipose tissue.

 


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  		Fig. 2B. —Coronal T1-weighted spin-echo MR images obtained with volume segmentation for total adipose tissue. Contiguous images of 30-week-old lysosomal acid lipase–deficient mouse show red regions of interest that denote area of total adipose tissue. Liver (L) is larger than that in control mouse. Decreased subcutaneous fat and increased fat deposition in bowel wall (arrows) are seen.

 


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  		Fig. 3A. —Coronal T1-weighted spin-echo MR images obtained with volume segmentation for intraabdominal adipose tissue. Contiguous images of 30-week-old control mouse show red regions of interest that denote area of intraabominal adipose tissue. Retroperitoneal (arrows) and reproductive (arrowheads) adipose tissue is seen.

 


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  		Fig. 3B. —Coronal T1-weighted spin-echo MR images obtained with volume segmentation for intraabdominal adipose tissue. Contiguous images of 30-week-old lysosomal acid lipase–deficient mouse show red regions of interest that denote area of intraabdominal adipose tissue. Retroperitoneal and reproductive adipose tissue is less than that in control mouse, and fat deposition in bowel wall (arrows) is greater than that in control mouse. Hepatomegaly is present also.

 


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  		Fig. 4A. —Coronal T1-weighted spin-echo MR images obtained with volume segmentation for liver. Contiguous images of 30-week-old control mouse show red regions of interest that denote area of liver.

 


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  		Fig. 4B. —Coronal T1-weighted spin-echo MR images obtained with volume segmentation for liver. Contiguous images of 30-week-old lysosomal acid lipase–deficient mouse show red regions of interest that denote area of liver. Liver is markedly larger in lysosomal acid lipase–deficient mouse than that in control mouse.

 


Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Multiple studies [3–15] have shown a strong relationship between increased visceral adiposity (increased IAT/SAT ratio) and the risk that cardiovascular disease and type 2 diabetes mellitus will develop. Although the precise mechanism by which increased intraabdominal fat increases cardiovascular risk is unknown, one hypothesis is that fat in the abdominal cavity is more metabolically active than fat in subcutaneous tissues. Such studies have shown a positive correlation between MRI parameters for visceral adiposity and other measures of cardiovascular risk, such as abnormalities of blood lipoprotein concentrations [1–14], high triglyceride concentrations, high circulating insulin levels, and an increased incidence of hypertension [1–14]. However, the accuracy of MRI techniques for evaluating visceral adiposity in living subjects has been limited because of the lack of a gold standard. A previous study did show that techniques were accurate in an in vitro phantom that used containers of dairy creamer and water to simulate abdominal adiposity [15]. Our study found no statistically significant differences between adipose tissue volumes calculated on MRI and those found on dissection at autopsy, further suggesting that MRI with volume segmentation analysis is accurate for measuring visceral adiposity.

Lysosomal acid lipase is a critical enzyme for hydrolyzing cholesteryl esters and triglycerides in lysosomes that are delivered by low-density-lipoprotein receptors and other receptor-mediated endocytosis pathways [16, 17]. Disruption of lysosomal acid lipase by gene targeting in the LAL-deficient mice resulted in the accumulation of cholesteryl esters and triglycerides in multiple organs, including the liver, spleen, small intestine, and adrenal glands [16, 17]. Age-dependent depletion of white and brown adipose tissues, lipodystrophy, and insulin resistance on glucose challenge also developed in the LAL-deficient mice. By the age of 6 months, LAL-deficient mice have almost no detectable white adipose tissue at fat deposits in the inguinal fat pad, retroperitoneal fat pad, and interscapular fat pads. The only remaining white adipose tissue is a reduced amount in the epididymis fat pad [16, 17]. Although the LAL-deficient and control mice maintain the same body weight, the distribution of fat is markedly different [16, 17]. This lipodystrophy phenotype provides a model system for monitoring adipose tissue changes in vivo using MRI.

In the comparison between LAL-deficient mice and control mice, statistically significant differences were found in the volume of adipose tissue within the reproductive tissues, in the liver volume, and in the IAT/SAT ratio. In LAL-deficient mice, wasting of normal stores of fat occurs within the subcutaneous tissues, retroperitoneum, reproductive area, and omentum, and fat accumulates within the liver, spleen, and small intestine. There are both striking differences in fat distribution between control mice and LAL-deficient mice and dramatic changes in fat distribution from birth to 6 months in LAL-deficient mice [16, 17].

LAL-deficient mice provide a model by which pathologic changes in visceral adiposity can be studied over time. MRI offers the advantage, over dissection of fat at autopsy, of being able to measure fat volumes sequentially over time. MRI of this model allows evaluation of the effect of therapeutic interventions, such as adenovirus-mediated gene therapy with LAL expression, on adipose volumes.

In conclusion, this study was preliminary and imaged few mice. However, LAL-deficient mice are a unique model with a pattern of adipose distribution both differing markedly from control mice and changing dramatically over time. MRI provides a tool to measure these changes in adipose distribution sequentially and may help in further research on the effect of therapeutic interventions in this model. In addition, comparison of volume measurements of adipose tissue on MRI and at autopsy further supports the accuracy of MRI in evaluating visceral adiposity.


References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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