Successful adaptation to ketosis by mice with tissue-specific deficiency of ketone body oxidation

1Washington University.
AJP Endocrinology and Metabolism (Impact Factor: 3.79). 12/2012; 304(4). DOI: 10.1152/ajpendo.00547.2012
Source: PubMed


During states of low carbohydrate intake, mammalian ketone body metabolism transfers energy substrates originally derived from fatty acyl chains within the liver to extrahepatic organs. We previously demonstrated that the mitochondrial enzyme CoA transferase (succinyl-CoA:3-oxoacid CoA transferase, SCOT, encoded by nuclear Oxct1) is required for oxidation of ketone bodies, and that germline SCOT-knockout (KO) mice die within 48h of birth due to hyperketonemic hypoglycemia. Here, we use novel transgenic and tissue-specific SCOT-KO mice to demonstrate that ketone bodies do not serve an obligate energetic role within highly ketolytic tissues during the ketogenic neonatal period or during starvation in the adult. While transgene-mediated restoration of myocardial CoA transferase in germline SCOT-KO mice is insufficient to prevent lethal hyperketonemic hypoglycemia in the neonatal period, mice lacking CoA transferase selectively within neurons, cardiomyocytes, or skeletal myocytes, are all viable as neonates. Like germline SCOT-KO neonatal mice, neonatal mice with neuronal CoA transferase deficiency exhibit increased cerebral glycolysis and glucose oxidation, and while these neonatal mice exhibit modest hyperketonemia, they do not develop hypoglycemia. As adults, tissue-specific SCOT-KO mice tolerate starvation, exhibiting only modestly increased hyperketonemia. Finally, metabolic analysis of adult germline Oxct1(+/-) mice demonstrates that global diminution of ketone body oxidation yields hyperketonemia, but hypoglycemia emerges only during a protracted state of low carbohydrate intake. Together, these data suggest that, at the tissue level, ketone bodies are not a required energy substrate in the newborn period or during starvation, but rather that integrated ketone body metabolism mediates adaptation to ketogenic nutrient states.

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    • "In the mitochondria, acetoacetate is activated by coenzyme A (CoA) transferase [succinyl-CoA:3-oxoacid CoA transferase (SCOT); EC] to acetoacetyl-CoA, which is then converted to acetylcoA and introduced into the tricarboxylic acid (TCA) cycle for energy production. SCOT knockout mice exhibit marked hyperketonemia, showing the importance of the appropriate regulation of ketone body metabolism during the neonatal period [3] [6]. "
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    ABSTRACT: Acetoacetyl-CoA synthetase (AACS) is a ketone body-utilizing enzyme, which is responsible for the synthesis of cholesterol and fatty acids from ketone bodies in lipogenic tissues, such as the liver and adipocytes. To explore the possibility of AACS regulation at the protein-processing level, we investigated the proteolytic degradation of AACS. Western blot analysis showed that the 75.1 kDa AACS was cleaved to form a protein of approximately 55 kDa in the kidney, which has considerable high activity of legumain, a lysosomal asparaginyl endopeptidase. Co-expression of AACS and legumain in HEK 293 cells generated the 55 kDa product from AACS. Moreover, incubation of recombinant AACS with recombinant legumain resulted in the degradation of AACS. Knockdown of legumain with short-hairpin RNA against legumain using the hydrodynamics method led to a decrease in the 55 kDa band of AACS in mouse kidney. These results suggest that legumain is involved in the processing of AACS through the lysosomal degradation pathway in the kidney.
    Preview · Article · Oct 2014 · Biochemical and Biophysical Research Communications
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    • "Animals and diets The Animal Studies Committee at Washington University approved all experiments prior to their performance. SCOT-Heart-KO mice were generated by successive generations of breeding of Oxct1 flox/flox mice to mice expressing Cre recombinase under control of the alpha myosin heavy chain promoter (aMHC-Cre, Jackson Laboratory, stock number 011038) [31]. SCOT-Heart-KO and aMHC-Cre control mice (male mice were studied) were maintained for at least ten generations on a C57BL/ 6N Â C57BL/6J hybrid substrain background. "
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    ABSTRACT: Objective: Exploitation of protective metabolic pathways within injured myocardium still remains an unclarified therapeutic target in heart disease. Moreover, while the roles of altered fatty acid and glucose metabolism in the failing heart have been explored, the influence of highly dynamic and nutritionally modifiable ketone body metabolism in the regulation of myocardial substrate utilization, mitochondrial bioenergetics, reactive oxygen species (ROS) generation, and hemodynamic response to injury remains undefined. Methods: Here we use mice that lack the enzyme required for terminal oxidation of ketone bodies, succinyl-CoA:3-oxoacid CoA transferase (SCOT) to determine the role of ketone body oxidation in the myocardial injury response. Tracer delivery in ex vivo perfused hearts coupled to NMR spectroscopy, in vivo high-resolution echocardiographic quantification of cardiac hemodynamics in nutritionally and surgically modified mice, and cellular and molecular measurements of energetic and oxidative stress responses are performed. Results: While germline SCOT-knockout (KO) mice die in the early postnatal period, adult mice with cardiomyocyte-specific loss of SCOT (SCOT-Heart-KO) remarkably exhibit no overt metabolic abnormalities, and no differences in left ventricular mass or impairments of systolic function during periods of ketosis, including fasting and adherence to a ketogenic diet. Myocardial fatty acid oxidation is increased when ketones are delivered but cannot be oxidized. To determine the role of ketone body oxidation in the remodeling ventricle, we induced pressure overload injury by performing transverse aortic constriction (TAC) surgery in SCOT-Heart-KO and αMHC-Cre control mice. While TAC increased left ventricular mass equally in both groups, at four weeks post-TAC, myocardial ROS abundance was increased in myocardium of SCOT-Heart-KO mice, and mitochondria and myofilaments were ultrastructurally disordered. Eight weeks post-TAC, left ventricular volume was markedly increased and ejection fraction was decreased in SCOT-Heart-KO mice, while these parameters remained normal in hearts of control animals. Conclusions: These studies demonstrate the ability of myocardial ketone metabolism to coordinate the myocardial response to pressure overload, and suggest that the oxidation of ketone bodies may be an important contributor to free radical homeostasis and hemodynamic preservation in the injured heart.
    Full-text · Article · Aug 2014 · Molecular Metabolism
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    • "This suggested that increased ketonemia in FATP1-mice is not due to upregulation of the ketogenic gene Hmgcs2 in skm. We thus reasoned that it could be due to regulation of OXCT1, a required enzyme for ketone body oxidation [52], which can be involved in ketogenesis [49]. We found that skm Oxct1 expression was enhanced by FATP1 in mice fed chow, but unchanged in those fed a high-fat diet, while OXCT1 protein levels were unchanged in both diet conditions. "
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    ABSTRACT: FATP1 mediates skeletal muscle cell fatty acid import, yet its intracellular localization and metabolic control role are not completely defined. Here, we examine FATP1 localization and metabolic effects of its overexpression in mouse skeletal muscle. The FATP1 protein was detected in mitochondrial and plasma membrane fractions, obtained by differential centrifugation, of mouse gastrocnemius muscle. FATP1 was most abundant in purified mitochondria, and in the outer membrane and soluble intermembrane, but not in the inner membrane plus matrix, enriched subfractions of purified mitochondria. Immunogold electron microscopy localized FATP1-GFP in mitochondria of transfected C2C12 myotubes. FATP1 was overexpressed in gastrocnemius mouse muscle, by adenovirus-mediated delivery of the gene into hindlimb muscles of newborn mice, fed after weaning a chow or high-fat diet. Compared to GFP delivery, FATP1 did not alter body weight, serum fed glucose, insulin and triglyceride levels, and whole-body glucose tolerance, in either diet. However, fatty acid levels were lower and β-hydroxybutyrate levels were higher in FATP1- than GFP-mice, irrespective of diet. Moreover, intramuscular triglyceride content was lower in FATP1- versus GFP-mice regardless of diet, and β-hydroxybutyrate content was unchanged in high-fat-fed mice. Electroporation-mediated FATP1 overexpression enhanced palmitate oxidation to CO2, but not to acid-soluble intermediate metabolites, while CO2 production from β-hydroxybutyrate was inhibited and that from glucose unchanged, in isolated mouse gastrocnemius strips. In summary, FATP1 was localized in mitochondria, in the outer membrane and intermembrane parts, of mouse skeletal muscle, what may be crucial for its metabolic effects. Overexpressed FATP1 enhanced disposal of both systemic fatty acids and intramuscular triglycerides. Consistently, it did not contribute to the high-fat diet-induced metabolic dysregulation. However, FATP1 lead to hyperketonemia, likely secondary to the sparing of ketone body oxidation by the enhanced oxidation of fatty acids.
    Full-text · Article · May 2014 · PLoS ONE
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