John R. Forder

Preferential inhibition of lactate oxidation relative to glucose oxidation in the rat heart following diabetes

OBJECTIVEnAlterations in myocardial metabolism occur early after the onset of diabetes suggesting that they may play a role in the development of cardiac dysfunction. Inhibition of myocardial pyruvate dehydrogenase (PDH), glucose transport and glycolysis have all been reported following diabetes. In vivo lactate is also a potential source of energy for the heart and its oxidation should not be affected by changes in glucose transport and glycolysis. Therefore, the objective of this study, was to test the hypothesis that following diabetes the inhibition of glucose oxidation would be greater than the inhibition of lactate oxidation.nnnMETHODSnHearts from control and one-week-old diabetic rats were perfused with [1-13C]glucose (11 mmol/l) alone, [1-13C]glucose plus lactate (0.5 mmol/l) or glucose plus [3-13C]lactate (0.5 or 1.0 mmol/l) as substrates. Glucose and lactate oxidation rates were determined by combining 13C-NMR glutamate isotopomer analysis of tissue extracts with measurements of oxygen consumption.nnnRESULTSnIn diabetic hearts perfused with glucose alone, glucose oxidation was decreased compared to controls (0.31 +/- 0.08 vs. 0.71 +/- 0.11 mumoles/min/g wet weight; p < 0.05). Surprisingly, in hearts perfused with glucose plus 0.5 mmol/l lactate, there was no difference in glucose oxidation between control and diabetic groups (0.20 +/- 0.05 vs. 0.16 +/- 0.04 mumoles/min/g wet weight respectively). However, under these conditions lactate oxidation was markedly reduced in the diabetic group (0.89 +/- 0.18 vs. 0.24 +/- 0.05 mumoles/min/g wet weight; p < 0.05). At 1.0 mmol/l lactate oxidation was still significantly depressed in the diabetic group.nnnCONCLUSIONnThere was a greater decrease in lactate oxidation relative to glucose oxidation in hearts from diabetic animals. These results demonstrate that diabetes leads to a specific inhibition of lactate oxidation independent of its effects on pyruvate dehydrogenase.

The heart in diabetes

1. Clinical Manifestations of Diabetic Cardiomyopathy F.S. Fein. 2. Pathological Alterations of the Heart in Diabetes Mellitus A. Borczuk, S.M. Factor. 3. Alterations of Cardiac Function, Composition and Rhythm as a Consequence of Diabetes M. Shanmugam, et al. 4. Therapeutic Interventions in the Diabetic Heart J.H. McNeill, B. Rodrigues. 5. Effect of Diabetes on Protein Synthesis in the Myocardium D. Geenen, A. Malhotra. 6. Remodelling of Subcellular Organelles During the Development of Diabetic Cardiomyopathy N.S. Dhalla, et al. 7. Ketone Body Metabolism in the Diabetic Heart J.R. Forder. 8. Cardiac Glycogen Metabolism in Diabetes M. Laughlin. 9. The Effect of Diabetes on Myocardial Glucose Metabolism J.C. Chatham. 10. Fatty Acid Metabolism in the Heart Following Diabetes G.D. Lopaschuk. 11. Conclusion J.C. Chatham, et al. Index.

Impact of 1 wk of diabetes on the regulation of myocardial carbohydrate and fatty acid oxidation

The aim of this study was to investigate the effect of increasing exogenous palmitate concentration on carbohydrate and palmitate oxidation in hearts from control and 1-wk diabetic rats. Hearts were perfused with glucose, [3-(13)C]lactate, and [U-(13)C]palmitate. Substrate oxidation rates were determined by combining (13)C-NMR glutamate isotopomer analysis of tissue extracts with measurements of oxygen consumption. Carbohydrate oxidation was markedly depressed after diabetes in the presence of low (0.1 mM) but not high (1.0 mM) palmitate concentration. Increasing exogenous palmitate concentration 10-fold resulted in a 7-fold increase in the contribution of palmitate to energy production in controls but only a 30% increase in the diabetic group. Consequently, at 0.1 mM palmitate, the rate of fatty acid oxidation was higher in the diabetic group than in controls; however, at 1.0 mM fatty acid oxidation, it was significantly depressed. Therefore, after 1 wk of diabetes, the major differences in carbohydrate and fatty acid metabolism occur primarily at low rather than high exogenous palmitate concentration.The aim of this study was to investigate the effect of increasing exogenous palmitate concentration on carbohydrate and palmitate oxidation in hearts from control and 1-wk diabetic rats. Hearts were perfused with glucose, [3-13C]lactate, and [U-13C]palmitate. Substrate oxidation rates were determined by combining13C-NMR glutamate isotopomer analysis of tissue extracts with measurements of oxygen consumption. Carbohydrate oxidation was markedly depressed after diabetes in the presence of low (0.1 mM) but not high (1.0 mM) palmitate concentration. Increasing exogenous palmitate concentration 10-fold resulted in a 7-fold increase in the contribution of palmitate to energy production in controls but only a 30% increase in the diabetic group. Consequently, at 0.1 mM palmitate, the rate of fatty acid oxidation was higher in the diabetic group than in controls; however, at 1.0 mM fatty acid oxidation, it was significantly depressed. Therefore, after 1 wk of diabetes, the major differences in carbohydrate and fatty acid metabolism occur primarily at low rather than high exogenous palmitate concentration.

Lactic acid and protein interactions: implications for the NMR visibility of lactate in biological systems

The addition of bovine serum albumin (BSA) to a solution of lactate and alanine resulted in the disappearance of the 1H-NMR resonances from lactate but not alanine. As temperature is increased lactate becomes increasingly NMR visible and after heating above 65 degreesC and cooling to 25 degreesC lactate binding is reduced. With a concentration of 0.2 mM BSA, there was a linear relationship between NMR visible lactate versus total lactate over a range of lactate concentrations of 0.2-35 mM (slope 0.384+/-0.003) indicating that approx. 60% of the added lactate is not visible in the 1H-NMR spectrum. With a 0.1 mM BSA solution, however, the slope was markedly higher indicating that under these conditions only 25-30% of the lactate was NMR invisible. The results from this study indicate that decreased NMR visibility of lactate in proteinaceous solutions is due to non-specific binding which is dependent on the tertiary structure of the protein. This has important implications not only for the interpretation of in vivo 1H-NMR experiments but also for 13C, and 14C studies of metabolism.

Acute L-triiodothyronine administration potentiates inotropic responses to β-adrenergic stimulation in the isolated perfused rat heart

OBJECTIVEnAcute inotropic effects of triiodothyronine (T3) have been reported, employing both in vivo experimental animal models and in vitro isolated heart perfusions. However, the mechanisms responsible for these acute inotropic effects remain unclear. The aim of this study, therefore, was to delineate the role of the beta-adrenergic receptor system in these acute responses.nnnMETHODSnThe hearts from both euthyroid and hypothyroid (treated with 0.05% PTU in drinking water) male Sprague-Dawley rats were used in 5 experimental study protocols. Hearts from euthyroid rats were perfused with buffer containing either T3(10(-7) M) or control while continuously recording left ventricular function for 10 min (acute effects). Two-hour perfusions (subacute effects) and cardiac responses following increasing doses of isoproterenol (10(-10) to 10(-6) M) in the presence or absence of T3-containing buffer (acute interaction) were also determined. In hypothyroid rats, the subacute responses and the acute interactions were investigated.nnnRESULTSnIn the presence of T3, an acute, significant potentiation of the inotropic responses following beta-adrenergic stimulation with isoproterenol was observed in both rat cohorts, which was more pronounced in hearts from euthyroid rats. An acute (< 40 s), but transient (79 +/- 8 s), direct inotropic response was observed in hearts from euthyroid rats. No cardiac responses were seen during a 2-h perfusion in hearts from either euthyroid or hypothyroid rats.nnnCONCLUSIONSnThe acute inotropic effects of T3 in non-ischemic myocardium probably result from an acute interaction between T3 and catecholamines rather than through a direct inotropic effect of T3 alone.

A Microperfusion and In-Bore Oxygenator System Designed for Magnetic Resonance Microscopy Studies on Living Tissue Explants

Spectrometers now offer the field strengths necessary to visualize mammalian cells but were not designed to accommodate imaging of live tissues. As such, spectrometers pose significant challenges—the most evident of which are spatial limitations—to conducting experiments in living tissue. This limitation becomes problematic upon trying to employ commercial perfusion equipment which is bulky and—being designed almost exclusively for light microscopy or electrophysiology studies—seldom includes MR-compatibility as a design criterion. To overcome problems exclusive to ultra-high magnetic field environments with limited spatial access, we have designed microperfusion and in-bore oxygenation systems capable of interfacing with Bruker’s series of micro surface-coils. These devices are designed for supporting cellular resolution imaging in MR studies of excised, living tissue. The combined system allows for precise control of both dissolved gas and pH levels in the perfusate thus demonstrating applicability for a wide range of tissue types. Its compactness, linear architecture, and MR-compatible material content are key design features intended to provide a versatile hardware interface compatible with any NMR spectrometer. Such attributes will ensure the microperfusion rig’s continued utility as it may be used with a multitude of contemporary NMR systems in addition to those which are currently in development.

Imaging of Cardiotoxicity

A driamycin (ADR; doxorubicin HCl), is a potent anthracycline chemotherapeutic agent that is effective against a wide range of human malignancies, such as leukemias, lymphomas, and many solid tumors. For many malignancies (eg, breast cancer, lymphoma), it is the most effective single agent. The use of ADR has been limited, however, by the development of a dose-dependent cardiomyopathy induced by this antineoplastic agent. The overall incidence of clinical cardiotoxicity for patients treated with ADR is approximately 2%, with the incidence of congestive heart failure increasing to 10% at 500 mg/m and to over 40% at 700 mg/m. The cardiac-specific injury of ADR is usually irreversible, although aggressive support for a low cardiac output may reverse the dysfunction in some patients. Nevertheless, once ADR-induced heart failure has been diagnosed, the prognosis is poor; mortality varies from 30 to 60%. It is also noteworthy that various other commonly used antineoplastic agents, cyclophosphamide, 5-fluorouracil, paclitaxel, streptozotocin, bevacizumab (Avastin), etoposide, bleomycin, tamoxifen, and trastuzumab (Herceptin), as well as radiation therapy, which is commonly used as an adjuvant to lumpectomy, are also cardiotoxic, although different mechanisms may be involved. Therefore, the development of sensitive methods for early detection of drugor radiation-induced cardiotoxicity in individual patients could greatly facilitate the management of a wide range of neoplastic diseases. Development of ADR-induced cardiomyopathy is dose dependent and cumulative. To reduce the incidence of cardiotoxicity, limiting treatment with this agent to a total lifetime dose of ADR of 550 mg/m is recommended. However, the individual response to ADR is highly variable, and bolus administration of up to 900 mg/m has been reported without evidence of heart failure; up to 1,500 mg/m has been administered by continuous infusion without evidence of cardiotoxicity. In some patients, drug resistance, rather than toxicity, is dose limiting; in other instances, restriction of ADR therapy to the recommended guidelines may lead to the premature discontinuation of treatment in patients who might otherwise benefit from higher doses. Conversely, some patients may be highly sensitive to ADR, experiencing toxicity at doses that are lower than the recommended limit. Patients with cardiac disorders or with previous radiation therapy to the mediastinum are generally excluded from ADR treatment protocols. It has also been reported that in some pediatric cases, for which treatment with ADR was well within the recommended limit, congestive heart failure developed several years after treatment. Consequently, the availability of a sensitive, noninvasive method for assessing the early onset of ADR cardiomyopathy in the individual patient would have a significant clinical impact. At the present time, there does not appear to be any effective method for predicting which patients will develop ADR-induced heart failure or for sensitively detecting its onset at a very early stage, when administration can effectively be discontinued or useful treatment for cardiomyopathy can be initiated. Current methods of assessing cardiotoxicity are limited to measurement of cardiac function by radionuclide ventriculography (RNV), echocardiography, and detection of tissue damage by percutaneous endomyocardial biopsy. RNV is not particularly sensitive for detecting early damage as assessed by endomyocardial biopsy scores. In combination with exercise, RNV sensitivity improves, but at the expense of specificity. Artifactual errors of 5% or more in ejection fraction measurements are common. Therefore, it has been recommended that a combination of RNV or echocardiography and biopsy examinations be employed in the From the Department of Radiology, University of Pennsylvania, Philadelphia, PA; Department of Radiology, University of Florida, Miami, FL; and Division of Cardiovascular Disease, Department of Medicine, University of Alabama in Birmingham, Birmingham, AL.

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes. While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissues specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.