Abstract
The Native American Pima population has the highest incidence of insulin resistance (IR) and type 2 diabetes mellitus (T2DM) of any reported population, but the pathophysiologic mechanism is unknown. Genetic studies in Pima Indians have linked acyl-CoA dehydrogenase 10 (ACAD10) gene polymorphisms, among others, to this predisposition. The gene codes for a protein with a C-terminus region that is structurally similar to members of a family of flavoenzymes—the acyl-CoA dehydrogenases (ACADs)—that catalyze α,β-dehydrogenation reactions, including the first step in mitochondrial FAO (FAO), and intermediary reactions in amino acids catabolism. Dysregulation of FAO and an increase in plasma acylcarnitines are recognized as important in the pathophysiology of IR and T2DM. To investigate the deficiency of ACAD10 as a monogenic risk factor for T2DM in human, an Acad-deficient mouse was generated and characterized. The deficient mice exhibit an abnormal glucose tolerance test and elevated insulin levels. Blood acylcarnitine analysis shows an increase in long-chain species in the older mice. Nonspecific variable pattern of elevated short-terminal branch-chain acylcarnitines in a variety of tissues was also observed. Acad10 mice accumulate excess abdominal adipose tissue, develop an early inflammatory liver process, exhibit fasting rhabdomyolysis, and have abnormal skeletal muscle mitochondria. Our results identify Acad10 as a genetic determinant of T2DM in mice and provide a model to further investigate genetic determinants for insulin resistance in humans.
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Abbreviations
- ACADs:
-
Acyl-CoA dehydrogenases
- ACAD10:
-
Acyl-CoA dehydrogenase 10
- ACNs:
-
Acylcarnitines
- BN-PAGE:
-
Blue native polyacrylamide gel electrophoresis°
- GWAS:
-
Genome-wide association study
- T2DM:
-
Type 2 diabetes mellitus
- FAO:
-
FAO
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Acknowledgements
Jonathan Franks and Ming Sun at the University of Pittsburgh’s Center for Biologic Imaging provided expertise in transmission electron microscopy. This work was supported in part by PHS Grant R01-DK 54936. All mouse experiments were approved by the University of Pittsburgh Animal Care and Use Committee.
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Supplemental Fig. 1
A. In a 5-week period, mouse, food levels from 6–7 mice per group were weighed weekly using a standard lab balance. All wild-type and Acad10-deficient mice were approximately 8 months old. The “WT pooled” and “Acad10-deficient pooled” categories include combined data from both male and female mice. There were no significant differences in the amount of food consumed by wild-type mice (any group) compared with Acad10-deficient mice (any group). B. Zones of movement quantitated in open-field testing. To examine locomotor function and behaviors of mutant mice in a new environment, animals were placed for 30 min in an open-field testing chamber (28 x 28 x 40 cm) with a floor divided into equal-sized square fields, and the frequency of a variety of behaviors was determined. The lighting was set to 15–20 lux, similar to standard housing. The animal was removed from its home cage and placed into one of the four corners of the open-field chamber and allowed to explore the chamber freely for 30 min, following which, it was returned to its home cage. Testing parameters included (1) ambulatory distance, (2) velocity, (3) real vertical time, (4) real stereotypic time, (5) distance per trip, (6) real resting time, (7) real ambulatory time, (8) ambulatory counts, (9) stereotypic counts, and (10) vertical counts. C. Distance traveled in each zone is depicted with wild-type animals in blue and ACAD10–/– deficient in red. The Acad10-deficient female mice show a statistically significant decrease in movement measures in Zone 1 (two-tailed p value=0.0004,0.0002, and 0.0003, respectively), indicated by an asterisk (*), compared with wild-type control mice of the same background. The Acad10-deficient female mice show statistically significant decrease in movement measures in Zone 0 across time (two-tailed p value=0.0230), indicated by an asterisk (*), compared with wild-type control mice of the same background. (PPTX 171 kb)
Supplemental Fig. 2
Mouse histopathology. A. Light microscopy with hematoxylin and eosin staining of wild-type liver (a) and ACAD10 –/– liver (b), and spleen (e) show the formation of small necroinflammatory lesions and early abscesses in liver, with extramedullary hematopoieisis and prominent megakaryocytes in spleen. B. Transmission electron microscopy of wild-type mouse liver (a) and muscle from hind quarter (c) was unremarkable. ACAD10-deficient liver (b) and muscle from hind quarter (d) show accumulation of lipid droplets (yellow arrows) and enlarged mitochondria (white arrow) in multiple representative sections. Muscle from hind quarter (d) also demonstrated abnormal shape and size of mitochondria in multiple representative sections. C. Specialized staining techniques of wild-type and deficient mouse tissues. a, b. Alkaline phosphatase staining shows peripheral nuclei indicative of chronic damage; c, d. Gomorri trichrome shows increased fat accumulation (purple) in deficient animals; e, f. Nicotinamide adenine dinucleotide, reduced (NADH) dehydrogenase staining shows an increased number of poorly staining muscle fibers. (PPTX 1049 kb)
Supplemental Fig. 3
Subcellular localization of ACAD10 varies in different tissues. A. Immunofluorescent staining of lung, pancreas, muscle, and kidney from wild-type mice with anitserum to ACAD10 (left column), the mitochondrial marker MTCO-1 (middle column), and the merged image (right column). All tissues showed colocalization of ACAD10 with the mitochondrial marker. B. In addition, lung and pancreas were stained with antibodies to the peroxisomal marker catalase (left column). Right column shows merged images, and identifies colocalization of ACAD10 to peroxisomes as well as mitochondria in lung and pancreas; ×60 all images. (PPTX 686 kb)
Supplemental Fig. 4
A. Hepatic phosphoenolpyruvate carboxykinase (PEPCK) expression and respiratory chain function. There is significant increase in hepatic expression of PEPCK in Acad10-deficient mice, which indicates hepatic insulin resistance. B. Blue native gel electrophoresis showing changes in expression of the complexes of the electron transport chain. Complex I activity is increased in liver, muscle, and brain. Complex V activity is increased in heart, liver, and muscle. (PPTX 196 kb)
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Bloom, K., Mohsen, AW., Karunanidhi, A. et al. Investigating the link of ACAD10 deficiency to type 2 diabetes mellitus. J Inherit Metab Dis 41, 49–57 (2018). https://doi.org/10.1007/s10545-017-0013-y
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DOI: https://doi.org/10.1007/s10545-017-0013-y