Diabetes Volume 67, June 2018 1057 Cardiac Dysfunction and Metabolic Inflexibility in a Mouse Model of Diabetes Without Dyslipidemia Maria Rohm,1 Dragana Savic,2 Vicky Ball,2 M. Kate Curtis,2 Sarah Bonham,3 Roman Fischer,3 Nathalie Legrave,4 James I. MacRae,4 Damian J. Tyler,2 and Frances M. Ashcroft1 Diabetes 2018;67:1057–1067 | https://doi.org/10.2337/db17-1195 Diabetes is a well-established risk factor for heart disease, and heart failure (1,2). Alterations in cardiac metabolism, leading to impaired cardiac function and a metabolic switch including changes in substrate utilization and mitochondrial toward fatty acid usage. In this study, we investigated if dysfunction, contribute to impaired heart function in di- hyperglycemia/hypoinsulinemia in the absence of dyslipi- abetes (3–6). The heart uses both glucose and free fatty acids demia is sufficient to drive these changes and if they can be (FFA) as fuel, with FFA accounting for 60–70% of energy reversed by restoring euglycemia. Using the bV59M mouse generation under normal conditions (7). FFA are metabo- model, in which diabetes can be rapidly induced and re- lized to acetyl-CoA by b-oxidation, and if FFA uptake exceeds versed, we show that stroke volume and cardiac output b-oxidation, as in obesity, this leads to accumulation of lipids were reduced within 2 weeks of diabetes induction. Flux and lipid metabolites, eventually causing lipotoxicity. By METABOLISM through pyruvate dehydrogenase was decreased, as mea- contrast, chronic hyperglycemia produces glucotoxicity, sured in vivo by hyperpolarized [1-13C]pyruvate MRS. causing the formation of reactive oxygen species and ad- Metabolomics showed accumulation of pyruvate, lactate, vanced glycation end products (6). Impaired cardiac function alanine, tricarboxyclic acid cycle metabolites, and branched- fi chain amino acids. Myristic and palmitoleic acid were de- is common to all types of diabetes and not con ned to creased. Proteomics revealed proteins involved in fatty acid individuals with diabetes with obesity or dyslipidemia. How- metabolism were increased, whereas those involved in glu- ever, the relative contributions of lipotoxicity and glucotox- cose metabolism decreased. Western blotting showed en- icity to cardiac dysfunction in diabetes remain unclear. hanced pyruvate dehydrogenase kinase 4 (PDK4) and Most studies to date have focused on dyslipidemia and uncoupling protein 3 (UCP3) expression. Elevated PDK4 shown that elevated serum FFA drive metabolic alterations and UCP3 and reduced pyruvate usage were present in the heart (6,8–10). The role of hyperglycemia has been less 24 h after diabetes induction. The observed effects were well investigated. However, the fact that the hypertrophic independent of dyslipidemia, as mice showed no evidence of cardiomyopathy and left ventricular dysfunction in a mouse elevated serum triglycerides or lipid accumulation in periph- model of lipodystrophy were ameliorated by lowering blood eral organs (including the heart). The effects of diabetes were glucose levels (11) suggests hyperglycemia may contribute to reversible, as glibenclamide therapy restored euglycemia, impaired cardiac function in diabetes. Furthermore, non- cardiac metabolism and function, and PDK4/UCP3 levels. obese patients with diabetes (including type 1 or monogenic diabetes) may also develop cardiac complications (12,13), Diabetes is an increasing health burden worldwide and supporting the idea that hyperglycemia alone is sufficient to a major risk factor for developing cardiovascular disease cause cardiac disease. Likewise, in a large study of individuals 1Department of Physiology, Anatomy and Genetics and OXION, University of M.R. is currently affiliated with the Institute for Diabetes and Cancer (IDC), Oxford, Oxford, U.K. Helmholtz Center Munich, Neuherberg, Germany; Joint Heidelberg-IDC 2Cardiac Metabolism Research Group, Department of Physiology, Anatomy and Translational Diabetes Program, Heidelberg University Hospital, Heidelberg, Genetics, University of Oxford, Oxford, U.K. Germany; Molecular Metabolic Control, Medical Faculty, Technical University 3Discovery Proteomics Facility, Target Discovery Institute, University of Oxford, of Munich, Munich, Germany; German Center for Diabetes Research, Neuherberg, Oxford, U.K. Germany. 4 The Francis Crick Institute, London, U.K. © 2018 by the American Diabetes Association. Readers may use this article as Corresponding author: Frances M. Ashcroft, [email protected]. long as the work is properly cited, the use is educational and not for profit, and the Received 3 October 2017 and accepted 12 March 2018. work is not altered. More information is available at http://www.diabetesjournals .org/content/license. This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-1195/-/DC1. 1058 Cardiac Dysfunction in a Eulipidemic Model of Diabetes Diabetes Volume 67, June 2018 with type 2 diabetes (T2D), a 1% increase in HbA1c was (Innovative Research of America) and under 2% isoflurane associated with an increased risk of heart failure, indepen- anesthesia. A schematic overview of the mouse experiments dent of obesity, suggesting hyperglycemia is an independent is given in Supplementary Fig. 1D. For tissue analysis, mice risk factor for cardiac dysfunction (14). were culled by cervical dislocation, and hearts were collected, In this study, we used an inducible mouse model of snap-frozen in liquid nitrogen, pulverized, and stored at human neonatal diabetes to explore the effects of chronic 280°C for later analysis. hyperglycemia/hypoinsulinemia in the absence of dyslipide- mia (15,16) (bV59M mice). Our mouse expresses an acti- Blood Metabolites Blood glucose levels were measured from the tail vein using vating mutation (Kir6.2-V59M) in the KATP channel specifically in pancreatic b-cells, which prevents glucose- a FreeStyle Lite device and FreeStyle Lite test strips (Abbott stimulated insulin secretion. It has several advantages Laboratories). Serum was obtained by incubating whole over other mouse models of diabetes. First, diabetes is bloodonicefor30minfollowedbycentrifugationat caused by a b-cell–specific genetic defect rather than a toxin 3,000g and 4°C for 30 min. It was then snap-frozen in liquid (e.g., streptozotocin) that may have deleterious effects in nitrogen for later analysis. Serum glucose was measured other tissues. Second, diabetes is not associated with obesity using a glucose (HK) kit (Sigma-Aldrich). Serum triglyceride or insulin resistance. Third, diabetes can be induced in adult (ab65336; Abcam), FFA (ab65341; Abcam), cholesterol (ab65390; Abcam), and insulin (10-1247-01; Mercodia) lev- life, precluding compensatory developmental changes. els were measured using the indicated kits. Fourth, diabetes is rapidly reversible by treatment with sulphonylurea drugs (e.g., glibenclamide), which close the Cine MRI open KATP channels (13). It should be recognized, however, Mice were imaged on an 11.7T MRI instrument (Bruker) as that this mouse is both hyperglycemic and hypoinsulinemic, previously described (17). Eight to 10 short-axis slices (slice as is the case in both type 1 diabetes and nonobese T2D. thickness, 1.0 mm; matrix size, 256 3 256; field of view, In this study, we show that plasma hyperglycemia (in 25.6 3 25.6 mm; echo time/repetition time, 1.43/4.6 ms; conjunction with hypoinsulinemia) is a major driver of flip angle, 17.5°; and number of averages, 4) were acquired impaired cardiac metabolism and function. Gene induction with a cine fast low-angle shot sequence (18). Left ventric- caused a substrate switch from glucose oxidation toward ular volumes were derived using the freehand draw function lactate production and increased fatty acid (FA) metabo- in ImageJ (National Institutes of Health). For each heart, left lism. This led to a reduced cardiac output and stroke volume. ventricular mass, ejection fraction, stroke volume, and car- Restoration of euglycemia by glibenclamide completely re- diac output were calculated. stored cardiac metabolism and function after 2 weeks of diabetes. The time-dependent deterioration of cardiac func- Hyperpolarized MRS tion correlated with reduced glucose flux through pyruvate Experiments were performed between 7 and 11 A.M. when 13 dehydrogenase (PDH) and increased expression of PDH mice were in the fed state. A total of 40 mg [1- C]pyruvate kinase 4 (PDK4) and uncoupling protein 3 (UCP3). (Sigma-Aldrich) doped with 15 mmol/L trityl radical (OXO63; GE Healthcare) and 3 mL Dotarem (1:50 dilution; Guerbet) – RESEARCH DESIGN AND METHODS was hyperpolarized in a prototype polarizer, with 20 30 min of microwave irradiation (19). The sample was subsequently Animals dissolved in a pressurized and heated alkaline solution, Animal studies were conducted in accordance with the U.K. containing 2.4 g/L sodium hydroxide and 100 mg/L EDTA Animals (Scientific Procedures) Act (1986) and local ethical dipotassium salt (Sigma-Aldrich), to yield a solution guidelines (Medical Research Council’sResponsibilityinthe of 80 mmol/L hyperpolarized sodium [1-13C]pyruvate Use of Animals in Medical Research, 1993). Mice hemi- with a polarization of 30%. A total of 200 mLwasinjected zygously expressing an inducible Kir6.2-V59M transgene 13 b b over 10 s via the tail vein. C MR pulse-acquire cardiac selectively in pancreatic -cells ( V59M mice) were gener- spectra were acquired over 60 s following injection of hyper- ated and transgene expression induced by tamoxifen in- polarized [1-13C]pyruvate