Glucose Metabolism as a Pre-clinical Biomarker for the Golden Retriever Model of Duchenne Muscular Dystrophy

Purpose Metabolic dysfunction in Duchenne muscular dystrophy (DMD) is characterized by reduced glycolytic and oxidative enzymes, decreased and abnormal mitochondria, decreased ATP, and increased oxidative stress. We analyzed glucose metabolism as a potential disease biomarker in the genetically homologous golden retriever muscular dystrophy (GRMD) dog with molecular, biochemical, and in vivo imaging. Procedures Pelvic limb skeletal muscle and left ventricle tissue from the heart were analyzed by mRNA profiling, qPCR, western blotting, and immunofluorescence microscopy for the primary glucose transporter (GLUT4). Physiologic glucose handling was measured by fasting glucose tolerance test (GTT), insulin levels, and skeletal and cardiac positron emission tomography/X-ray computed tomography (PET/CT) using the glucose analog 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG). Results MRNA profiles showed decreased GLUT4 in the cranial sartorius (CS), vastus lateralis (VL), and long digital extensor (LDE) of GRMD vs. normal dogs. QPCR confirmed GLUT4 downregulation but increased hexokinase-1. GLUT4 protein levels were not different in the CS, VL, or left ventricle but increased in the LDE of GRMD vs. normal. Microscopy revealed diffuse membrane expression of GLUT4 in GRMD skeletal but not cardiac muscle. GTT showed higher basal glucose and insulin in GRMD but rapid tissue glucose uptake at 5 min post-dextrose injection in GRMD vs. normal/carrier dogs. PET/ CT with [18F]FDG and simultaneous insulin stimulation showed a significant increase (p = 0.03) in mean standard uptake values (SUV) in GRMD skeletal muscle but not pelvic fat at 5 min post-[18F]FDG /insulin injection. Conversely, mean cardiac SUV was lower in GRMD than carrier/normal (p < 0.01). Conclusions Altered glucose metabolism in skeletal and cardiac muscle of GRMD dogs can be monitored with molecular, biochemical, and in vivo imaging studies and potentially utilized as a biomarker for disease progression and therapeutic response. Electronic supplementary material The online version of this article (10.1007/s11307-018-1174-2) contains supplementary material, which is available to authorized users.

Muscle samples were taken by surgical biopsy or at necropsy from the LDE, CS, VL, cranial tibial, diaphragm, and left ventricle of the heart; muscles from a combination of seven normal and nine GRMD samples were analyzed. Samples were imaged with an Olympus (Waltham, MA) FV1000 laser scanning confocal microscope using 20x/0.85 and 100x/1.4 oil immersion objectives. Images were examined with Olympus Fluoview FV1000 software. GLUT4 antibody (ab65267), Anti-SPTBN1 (ab72239), and goat anti-mouse IgG H&L (Alexa Fluor 488; ab 150113) were from Abcam. ImageJ (NIH; Bethesda, MD) 1.47v software was used for quantification of GLUT4 expression at the myofiber membrane in GRMD and normal muscle samples. Bitmap images taken at 20X objective were uploaded. For skeletal muscle analysis, a software-enabled standard default threshold was set for each image in color red with a dark background. The unit of length was set to "micron". Fluorescent particles were analyzed with measurements ranging from 50 microns 2 to infinity. The area and mean fluorescence of all particles were summed and the normalized fluorescence was calculated per image (summed area/summed fluorescence). Normalized values for all GRMD and normal muscle images were compared. Since cardiac muscle fibers differs in morphology, we could not use the same methodology as for skeletal muscle. As such, we measured the mean and peak intensity of GLUT-4 signal across the cardiomyocyte membrane.
An average of 10 cells per image for the mean and peak intensities were used per sample. GRMD was then compared to normal.
Intravenous (IV) glucose tolerance test (GTT): Dogs were fasted for 15 h prior to testing. Area under curve was calculated for glucose and insulin, as described previously [5]. Weights were taken the day of the procedure and catheters placed in both cephalic veins. After baseline glucose and insulin testing (0 min), each dog received a bolus of 50% dextrose solution intravenously (IV) at 1 g/kg body weight over 30 s. The opposite catheter was used for blood sampling. Three ml of blood were collected at 0 (pre-dextrose administration), 5, 15, 30, 45, 60, and 120 min postdextrose administration. The samples were centrifuged within 30 min and the serum frozen at -  [6]. Blood glucose was checked every 30 min. We took precautions to keep BG in the normal range (80-210 mg/dl) to prevent hypoglycemia, but also to prevent hyperglycemia, as the latter has been shown to interfere with radionuclide uptake. As such, BG levels were maintained between 70-100 mg/dl. A 10ml bolus of 50% dextrose was given IV if BG dropped below 80 mg/dl. The PET-CT procedure was performed on a 128-slice Siemens Biograph PET/CT scanner (Siemens Medical Solutions USA, Inc., Malvern, Pennsylvania). Dogs were placed in sternal recumbence with the pelvic limbs extended behind them. They were scanned caudal to cranial beginning two-thirds down the tibia (closer to the hock than the stifle) and progressing to the head with ~20 cm steps, 5 min/step, for seven steps total. A gated cardiac scan of the chest followed each full-body scan. The topogram prescription scout for the initial scan and the initial pair of PET scans began 5 min after [ 18 F]FDG and insulin administration, with a second set of scans at 1 h post administration.

Segmentation and analysis: PET-CT images were analyzed using the Inveon Research
Workspace (Siemens Medical Solutions USA, Inc., Malvern, Pennsylvania). For the skeletal muscle analysis, the full body scans were reoriented so that the transverse view was perpendicular to the long axis of the femur and the sagittal and dorsal views were parallel to the sagittal and frontal long axes of the femur. Using the CT scout scan, the muscles were manually segmented at every 5 th slice, and the whole muscle region of interest (ROI) was interpolated from these slices [7]. As our functional studies suggest that GRMD disease severity For the cardiac studies, all measurements were obtained using the 4 th bin of the gated cardiac scans. Scans were first reoriented such that the transverse, sagittal, and dorsal planes corresponded to the short axis, horizontal long axis (4 chamber), and the vertical long axis (2 chamber) views, respectively. The hearts were analyzed with three methods: 1) a single spherical ROI was placed over the whole left ventricle (LV) to record the max SUV; 2) the LV wall ROI was drawn: the window for PET was adjusted until the view showed an even uptake in the LV, then the slices in the LV were counted on the vertical long axis view; hearts ranged from 59 to 65 slices. Segmentation excluded the apical 5 slices and the first 10 basal slices to avoid overlapping structures that might otherwise alter results. Mean SUV, max SUV, and SD of SUV were recorded for each dog. To obtain a heterogeneity index, coefficient of variance (CoV) was calculated by dividing SD SUV by mean SUV [8]; and 3) to examine regional wall segments within the LV, 16 individual ROI's within each heart were examined [9,10]. The 16 ROIs were obtained by segmenting representative basal, mid-cavity, and apical slices for each heart as described in the American Heart Association scientific statement on standardized myocardial segmentation [11]; the true apex could not be reliably identified so was not evaluated.
Statistics: Statistics were performed in both Excel for Mac (Microsoft Corporation, Redmond, WA) and confirmed in Prism software version 6 (GraphPad Software, La Jolla, CA). The Prism software was also used for schematic graph generation. Two-way ANOVAs were employed to test significance for mean and max SUV between genotypes (GRMD, carrier, and normal) and muscle (CS, VL, LDE). ANOVAs were also used to look at differences in SUV for the regional segments in the heart. Non-parametric t-tests (Wilcoxon rank sum tests) were used to test significance of GLUT4 expression and CoV between GRMD, carrier and normal dogs. Significance was defined as p < .05.