Metabolic Profiling of the Diabetic Heart: Toward a Richer Picture

The increasing global prevalence of diabetes has been accompanied by a rise in diabetes-related conditions. This includes diabetic cardiomyopathy (DbCM), a progressive form of heart disease that occurs with both insulin-dependent (type-1) and insulin-independent (type-2) diabetes and arises in the absence of hypertension or coronary artery disease. Over time, DbCM can develop into overt heart failure. Like other forms of cardiomyopathy, DbCM is accompanied by alterations in metabolism which could lead to further progression of the pathology, with metabolic derangement postulated to precede functional changes in the diabetic heart. Moreover in the case of type-2 diabetes, underlying insulin resistance is likely to prevent the canonical substrate switch of the failing heart away from fatty acid oxidation toward increased use of glycolysis. Analytical chemistry techniques, collectively known as metabolomics, are useful tools for investigating the condition. In this article, we provide a comprehensive review of those studies that have employed metabolomic techniques, namely chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy, to profile metabolic remodeling in the diabetic heart of human patients and animal models. These studies collectively demonstrate that glycolysis and glucose oxidation are suppressed in the diabetic myocardium and highlight a complex picture regarding lipid metabolism. The diabetic heart typically shows an increased reliance on fatty acid oxidation, yet triacylglycerols and other lipids accumulate in the diabetic myocardium indicating probable lipotoxicity. The application of lipidomic techniques to the diabetic heart has identified specific lipid species that become enriched and which may in turn act as plasma-borne biomarkers for the condition. Metabolomics is proving to be a powerful approach, allowing a much richer analysis of the metabolic alterations that occur in the diabetic heart. Careful physiological interpretation of metabolomic results will now be key in order to establish which aspects of the metabolic derangement are causal to the progression of DbCM and might form the basis for novel therapeutic intervention.

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title: Determination of triglyceride in the human myocardium by magnetic resonance spectroscopy: reproducibility and sensitivity of the method.
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title: Exercise-induced modulation of cardiac lipid content in healthy lean young men
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title: Myocardial phosphocreatine-to-ATP ratio is a predictor of mortality in patients with dilated cardiomyopathy.
abstract: In patients with heart failure due to dilated cardiomyopathy, cardiac energy metabolism is impaired, as indicated by a reduction of the myocardial phosphocreatine-to-ATP ratio, measured noninvasively by 31P-MR spectroscopy. The purpose of this study was to test whether the phosphocreatine-to-ATP ratio also offers prognostic information in terms of mortality prediction as well as how this index compares with well-known mortality predictors such as left ventricular ejection fraction (LVEF) or New York Heart Association (NYHA) class.



Thirty-nine patients with dilated cardiomyopathy were followed up for 928+/-85 days (2.5 years). At study entry, LVEF and NYHA class were determined, and the cardiac phosphocreatine-to-ATP ratio was measured by localized 31P-MR spectroscopy of the anterior myocardium. During the study period, total mortality was 26%. Patients were divided into two groups, one with a normal phosphocreatine-to-ATP ratio (>1.60; mean+/-SE, 1.98+/-0.07; n=19; healthy volunteers: 1.94+/-0.11, n=30) and one with a reduced phosphocreatine-to-ATP ratio (<1.60; 1.30+/-0.05; n=20). At re-evaluation (mean, 2.5 years), 8 of 20 patients with reduced phosphocreatine-to-ATP ratios had died, all of cardiovascular causes (total and cardiovascular mortality, 40%). Of the 19 patients with normal phosphocreatine-to-ATP ratios, 2 had died (total mortality, 11%), one of cardiovascular causes (cardiovascular mortality, 5%). Kaplan-Meier analysis showed significantly reduced total (P=.036) and cardiovascular (P=.016) mortality for patients with normal versus patients with low phosphocreatine-to-ATP ratios. A Cox model for multivariate analysis showed that the phosphocreatine-to-ATP ratio and NYHA class offered significant independent prognostic information on cardiovascular mortality.



The myocardial phosphocreatine-to-ATP ratio, measured noninvasively with 31P-MR spectroscopy, is a predictor of both total and cardiovascular mortality in patients with dilated cardiomyopathy.

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title: Real‐time assessment of Krebs cycle metabolism using hyperpolarized C magnetic resonance spectroscopy
abstract: The Krebs cycle plays a fundamental role in cardiac energy production and is often implicated in the energetic imbalance characteristic of heart disease. In this study, we measured Krebs cycle flux in real time in perfused rat hearts using hyperpolarized magnetic resonance spectroscopy (MRS). [2‐13C]Pyru‐ vate was hyperpolarized and infused into isolated perfused hearts in both healthy and postischemic metabolic states. We followed the enzymatic conversion of pyruvate to lactate, acetylcarnitine, citrate, and glutamate with 1 s temporal resolution. The appearance of 13C‐labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of 13C‐ labeled citrate and glutamate was decreased postischemia, as opposed to lactate, which was significantly elevated. These results showed that the control and fluxes of the Krebs cycle in heart disease can be studied using hyperpolarized [2‐13C]pyruvate.— Schroeder, M. A., Atherton, H. J., Ball, D. R., Cole, M. A., Heather, L. C., Griffin, J. L., Clarke, K., Radda, G. K., Tyler, D. J. Real‐time assessment of Krebs cycle metabolism using hyperpolarized 13C magnetic resonance spectroscopy. FASEBJ. 23, 2529–2538 (2009)

title: Cardiac metabolism measured noninvasively by hyperpolarized 13C MRI
abstract: Pyruvate is included in the energy production of the heart muscle and is metabolized into lactate, alanine, and CO2 in equilibrium with HCO  3− . The aim of this study was to evaluate the feasibility of using 13C hyperpolarization enhanced MRI to monitor pyruvate metabolism in the heart during an ischemic episode. The left circumflex artery of pigs (4 months, male, 29–34 kg) was occluded for 15 or 45 min followed by 2 hr of reperfusion. Pigs were examined by 13C chemical shift imaging following intravenous injection of 1‐13C pyruvate. 13C chemical shift MR imaging was used in order to visualize the local concentrations of the metabolites. After a 15‐min occlusion (no infarct) the bicarbonate signal level in the affected area was reduced (25–44%) compared with the normal myocardium. Alanine signal level was normal. After a 45‐min occlusion (infarction) the bicarbonate signal was almost absent (0.2–11%) and the alanine signal was reduced (27–51%). Due to image‐folding artifacts the data obtained for lactate were inconclusive. These studies demonstrate that cardiac metabolic imaging with hyperpolarized 1‐13C‐pyruvate is feasible. The changes in concentrations of the metabolites within a minute after injection can be detected and metabolic maps constructed.  2008.

title: Validation of the in vivo assessment of pyruvate dehydrogenase activity using hyperpolarised 13C MRS
abstract: Many diseases of the heart are characterised by changes in substrate utilisation, which is regulated in part by the activity of the enzyme pyruvate dehydrogenase (PDH). Consequently, there is much interest in the in vivo evaluation of PDH activity in a range of physiological and pathological states to obtain information on the metabolic mechanisms of cardiac diseases. Hyperpolarised [1‐13C]pyruvate, detected using MRS, is a novel technique for the noninvasive evaluation of PDH flux. PDH flux has been assumed to directly reflect in vivo PDH activity, although to date this assumption remains unproven. Control animals and animals undergoing interventions known to modulate PDH activity, namely high fat feeding and dichloroacetate infusion, were used to investigate the relationship between in vivo hyperpolarised MRS measurements of PDH flux and ex vivo measurements of PDH enzyme activity (PDHa). Further, the plasma concentrations of pyruvate and other important metabolites were evaluated following pyruvate infusion to assess the metabolic consequences of pyruvate infusion during hyperpolarised MRS experiments. Hyperpolarised MRS measurements of PDH flux correlated significantly with ex vivo measurements of PDHa, confirming that PDH activity influences directly the in vivo flux of hyperpolarised pyruvate through cardiac PDH. The maximum plasma concentration of pyruvate reached during hyperpolarised MRS experiments was approximately 250 µM, equivalent to physiological pyruvate concentrations reached during exercise or with dietary interventions. The concentrations of other metabolites, including lactate, glucose and β‐hydroxybutyrate, did not vary during the 60 s following pyruvate infusion. Hence, during the 60‐s data acquisition period, metabolism was minimally affected by pyruvate infusion.

title: Preparative gas chromatography and its applications.
abstract: Although hundreds of papers related to preparative gas chromatography (pGC) have been published since the late 1950s, the success of the GC technique has largely been associated with analytical instead of preparative purposes. Actually, pGC is an ideal alternative technique for the preparation of pure substances, especially volatile compounds. This paper reviews the papers (written in English) associated with pGC published over the period from the 1950s to the 2010s. For large scale preparation, large sample injection and vaporization, a high loading capacity column, a gas splitter at the end of the column and a special collecting device are fundamentally important for a pGC system. The primary components of pGC system, including injector, column, splitter, detector and collection traps, are briefly introduced. Furthermore, the applications of pGC in the separation and purification of volatile compounds from natural essential oils, in addition to the purification of isotopes, isomers and enantiomers are summarized.

title: Rodent models of diabetic cardiomyopathy
abstract: Diabetic cardiomyopathy increases the risk of heart failure in individuals with diabetes, independently of co-existing coronary artery disease and hypertension. The underlying mechanisms for this cardiac complication are incompletely understood. Research on rodent models of type 1 and type 2 diabetes, and the use of genetic engineering techniques in mice, have greatly advanced our understanding of the molecular mechanisms responsible for human diabetic cardiomyopathy. The adaptation of experimental techniques for the investigation of cardiac physiology in mice now allows comprehensive characterization of these models. The focus of the present review will be to discuss selected rodent models that have proven to be useful in studying the underlying mechanisms of human diabetic cardiomyopathy, and to provide an overview of the characteristics of these models for the growing number of investigators who seek to understand the pathology of diabetes-related heart disease.

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abstract: The focus of this overview is on the animal models of obesity most commonly utilized in research. The models include monogenic models in the leptin pathway, polygenic diet‐dependent models, and, in particular for their historical perspective, surgical and chemical models of obesity. However, there are far too many models to consider all of them comprehensively, especially those caused by selective molecular genetic approaches modifying one or more genes in specific populations of cells. Further, the generation and use of inducible transgenic animals (induced knock‐out or knock‐in) is not covered, even though they often carry significant advantages compared to traditional transgenic animals, e.g., influences of the genetic modification during the development of the animals can be minimized. The number of these animal models is simply too large to be covered in this unit.

title: Leptin- and Leptin Receptor-Deficient Rodent Models: Relevance for Human Type 2 Diabetes
abstract: Among the most widely used animal models in obesity-induced type 2 diabetes mellitus (T2DM) research are the congenital leptin- and leptin receptor-deficient rodent models. These include the leptin-deficient ob/ob mice and the leptin receptor-deficient db/db mice, Zucker fatty rats, Zucker diabetic fatty rats, SHR/N-cp rats, and JCR:LA-cp rats. After decades of mechanistic and therapeutic research schemes with these animal models, many species differences have been uncovered, but researchers continue to overlook these differences, leading to untranslatable research. The purpose of this review is to analyze and comprehensively recapitulate the most common leptin/leptin receptor-based animal models with respect to their relevance and translatability to human T2DM. Our analysis revealed that, although these rodents develop obesity due to hyperphagia caused by abnormal leptin/leptin receptor signaling with the subsequent appearance of T2DM-like manifestations, these are in fact secondary to genetic mutations that do not reflect disease etiology in humans, for whom leptin or leptin receptor deficiency is not an important contributor to T2DM. A detailed comparison of the roles of genetic susceptibility, obesity, hyperglycemia, hyperinsulinemia, insulin resistance, and diabetic complications as well as leptin expression, signaling, and other factors that confound translation are presented here. There are substantial differences between these animal models and human T2DM that limit reliable, reproducible, and translatable insight into human T2DM. Therefore, it is imperative that researchers recognize and acknowledge the limitations of the leptin/leptin receptor-based rodent models and invest in research methods that would be directly and reliably applicable to humans in order to advance T2DM management.

title: Animal models of metabolic syndrome: a review
abstract: Metabolic syndrome (MetS) consists of several medical conditions that collectively predict the risk for cardiovascular disease better than the sum of individual conditions. The risk of developing MetS in human depends on synergy of both genetic and environmental factors. Being a multifactorial condition with alarming rate of prevalence nowadays, establishment of appropriate experimental animal models mimicking the disease state in humans is crucial in order to solve the difficulties in evaluating the pathophysiology of MetS in human. This review aims to summarize the underlying mechanisms involved in the pathophysiology of dietary, genetic, and pharmacological models of MetS. Furthermore, we will discuss the usefulness, suitability, pros and cons of these animal models. Even though numerous animal models of MetS have been established, further investigations on the invention of new animal model and clarification of plausible mechanisms are still necessary to confer a better understanding to researchers on the selection of animal models for their studies.

title: Molecular mapping of the mouse ob mutation.
abstract: The mouse ob mutation has been mapped relative to a series of RFLPs among the progeny of three separate mouse crosses: an intraspecific backcross, an intraspecific intercross, and an interspecific intercross. Genotypic assignment at the ob locus was made by making use of measurements of body mass index and the plasma concentrations of glucose and insulin. These data have suggested that the development of diabetes in these animals is a consequence of unlinked polygenes. There was also evidence that unlinked Mus spretus alleles can diminish the obesity of ob/ob mice. From these data we have mapped several markers on chromosome 6 with the following order: cen-Cola-2-Met-ob-Cpa-Tcrb. The homologs of markers that flank ob map to human chromosome 7q, suggesting that if there is a human homologue of ob, it maps to 7q31.

title: Positional cloning of the mouse obese gene and its human homologue
abstract: The mechanisms that balance food intake and energy expenditure determine who will be obese and who will be lean. One of the molecules that regulates energy balance in the mouse is the obese (ob) gene. Mutation ofobresults in profound obesity and type II diabetes as part of a syndrome that resembles morbid obesity in humans. The ob gene product may function as part of a signalling pathway from adipose tissue that acts to regulate the size of the body fat depot.

title: Diabetes, a New Mutafton in the Mouse
abstract: Diabetes (db), which occurred in an inbred strain of mouse, is inherited as a unit autosomal recessive and is characterized by a metabolic disturbance resembling diabetes mellitus in man. Abnormal deposition of fat at 3 to 4 weeks of age is followed shortly by hyperglycemia, polyuria, and glycosuria. Accompanying morphological changes in the islets of Langerhans suggest neogenesis to compensate for insulin depletion.

title: Evidence That the Diabetes Gene Encodes the Leptin Receptor: Identification of a Mutation in the Leptin Receptor Gene in db/db Mice
abstract: OB-R is a high affinity receptor for leptin, an important circulating signal for the regulation of body weight. We identified an alternatively spliced transcript that encodes a form of mouse OB-R with a long intracellular domain. db/db mice also produce this alternatively spliced transcript, but with a 106 nt insertion that prematurely terminates the intracellular domain. We further identified G --> T point mutation in the genomic OB-R sequence in db/db mice. This mutation generates a donor splice site that converts the 106 nt region to a novel exon retained in the OB-R transcript. We predict that the long intracellular domain form of OB-R is crucial for initiating intracellular signal transduction, and as a corollary, the inability to produce this form of OB-R leads to the severe obese phenotype found in db/db mice.

title: Insulin and obesity in the Zucker genetically obese rat "fatty".
abstract: Insulin status and functional activity have been studied during the life of the Zucker “fatty” rat (an obese mutant). Serum immunoreactive insulin (IRI) is normal at 2 weeks when fattening is beginning, rises to a peak of 400 μ-U/ml at 15 weeks while fattening proceeds rapidly, then drops to near 200 μU/ml in older rats. This was observed with two normal diets and a high protein diet. When fattening is reversed by fasting adult “fatties,” IRI remains abnormally high until body lipid has returned to normal (79 days). Blood sugar, basally and after a glucose load, is normal in young and old “fatties,” and is barely above normal at peak IRI. Sugar falls to below 50 mg/100 ml with prolonged fasting. Amino nitrogen tends to be slightly low, free fatty acids (FFA) are very high, in “fatties.” There is a developing resistance of diaphragm to glucose uptake in vivo, both basally and after injected insulin. The role of elevated FFA in this resistance to glucose uptake by muscle is not known. Basal glucose uptake i...

title: Diabetes in Zucker diabetic fatty rat.
abstract: Male Zucker diabetic fatty fa/fa (ZDF) rats develop obesity and insulin resistance at a young age, and then with aging, progressively develop hyperglycemia. This hyperglycemia is associated with impaired pancreatic β-cell function, loss of pancreatic β-cell mass, and decreased responsiveness of liver and extrahepatic tissues to the actions of insulin and glucose. Of particular interest are the insights provided by studies of these animals into the mechanism behind the progressive impairment of carbohydrate metabolism. This feature among others, including the development of obesity- and hyperglycemia-related complications, is common between male ZDF rats and humans with type 2 diabetes associated with obesity. We discuss the diabetic features and complications found in ZDF rats and why these animals are widely used as a genetic model for obese type 2 diabetes.

title: Cardiovascular Changes in Animal Models of Metabolic Syndrome
abstract: Metabolic syndrome has been defined as a group of risk factors that directly contribute to the development of cardiovascular disease and/or type 2 diabetes. Insulin resistance seems to have a fundamental role in the genesis of this syndrome. Over the past years to the present day, basic and translational research has used small animal models to explore the pathophysiology of metabolic syndrome and to develop novel therapies that might slow the progression of this prevalent condition. In this paper we discuss the animal models used for the study of metabolic syndrome, with particular focus on cardiovascular changes, since they are the main cause of death associated with the condition in humans.

title: A Novel Locus, Mody4, Distal to D7Mit189 on Chromosome 7 Determines Early-Onset NIDDM in Nonobese C57BL/6 (Akita) Mutant Mice
abstract: In this article, we report on a nonobese C57BL/6 (B6) mouse model of NIDDM named Akita mouse, characterized by early age onset and autosomal dominant mode of inheritance. At 7 weeks of age, the mean morning blood glucose levels (mmol/1) under ad libitum feeding conditions were significantly higher (P < 0.01, analysis of variance [ANOVA]) in diabetic mice than in unaffected mice: 27.3 ± 5.3 for diabetic males (n = 50) and 9.3 ± 1.2 for unaffected males (n = 50); 13.6 ± 3.8 for diabetic females (n = 50) and 8.7 ± 1.1 for unaffected females (n = 50), while corresponding immunoreactive insulin levels in plasma were significantly lower in diabetic mice than in unaffected mice. In vitro insulin secretion was also impaired, even at 4 weeks of age. The 50% survival time for male diabetic mice (305 days) was significantly shorter than that of unaffected counterpart mice but not for diabetic females. Obesity did not occur in diabetic mice. Histological examinations of the pancreas in diabetic mice, from 4 to 35 weeks of age, revealed decreases in the numbers of active β-cells without insulitis. Morphometry demonstrated specific decreases in immunologically detectable insulin density in islets in diabetic mice, even at 4 weeks of age, without changes of relative islet areas. By linkage analysis, a single locus was identified on the basis of 178 N2 mice [(B6 × C3H/He)F1 × B6 and (B6 × C3H/He)F1 × C3H/He]. This locus, which we named Mody4, was mapped to chromosome 7 in a region 2–8 cM distal to D7Mitl89 (logarithm of odds [LOD] score = 15.6 and 10.3).

title: A mutation in the insulin 2 gene induces diabetes with severe pancreatic beta-cell dysfunction in the Mody mouse.
abstract: The mouse autosomal dominant mutation Mody develops hyperglycemia with notable pancreatic beta-cell dysfunction. This study demonstrates that one of the alleles of the gene for insulin 2 in Mody mice encodes a protein product that substitutes tyrosine for cysteine at the seventh amino acid of the A chain in its mature form. This mutation disrupts a disulfide bond between the A and B chains and can induce a drastic conformational change of this molecule. Although there was no gross defect in the transcription from the wild-type insulin 2 allele or two alleles of insulin 1, levels of proinsulin and insulin were profoundly diminished in the beta cells of Mody mice, suggesting that the number of wild-type (pro)insulin molecules was also decreased. Electron microscopy revealed a dramatic reduction of secretory granules and a remarkably enlarged lumen of the endoplasmic reticulum. Little proinsulin was processed to insulin, but high molecular weight forms of proinsulin existed with concomitant overexpression of BiP, a molecular chaperone in the endoplasmic reticulum. Furthermore, mutant proinsulin expressed in Chinese hamster ovary cells was inefficiently secreted, and its intracellular fraction formed complexes with BiP and was eventually degraded. These findings indicate that mutant proinsulin was trapped and accumulated in the endoplasmic reticulum, which could induce beta-cell dysfunction and account for the dominant phenotype of this mutation.

title: Genetic control of diabetes and insulitis in the nonobese diabetic (NOD) mouse
abstract: Genetic analysis of the development of diabetes and insulitis has been performed in the nonobese diabetic (NOD) mouse strain, a model of insulin-dependent (type I) diabetes mellitus. (NOD X C57BL/10)F1, F2, and (F1 X NOD) first-, second-, and third-backcross generations were studied. The data obtained were consistent with the hypothesis that diabetes is controlled by at least three functionally recessive diabetogenic genes, or gene complexes, one of which is linked to the MHC of the NOD. In contrast, pancreatic inflammation leading to insulitis was found to be controlled by a single incompletely dominant gene. One of the two diabetogenic loci that is not linked to the MHC appears to be essential for the development of severe insulitis. This diabetogenic gene may be identical to the gene that controls the initiation of the autoimmune response that progresses to insulitis. Although this gene appears to be functionally recessive in its control of diabetes, it is incompletely dominant in its control of insulitis. The MHC-linked diabetogenic gene, although not required for the development of insulitis, apparently influences the progression of the autoimmune response since NOD MHC homozygotes in the backcross generations displayed the highest incidence and most severe cases of insulitis. Interestingly, we have found two MHC heterozygous backcross females that have become diabetic, suggesting that either the MHC- linked diabetogenic gene is not strictly recessive or that a recombination event has occurred between the diabetogenic gene and the K or I-A regions of the MHC. The third diabetogenic locus appears to influence the progression of severe insulitis to overt diabetes. In animals homozygous at this locus, diabetes may result from a decreased ability to develop a protective suppressor response to the autoimmune process.

title: Genetic and pathogenic basis of autoimmune diabetes in NOD mice.
abstract: Non-obese diabetic (NOD) mice are an excellent model of T-cell mediated autoimmune insulin-dependent diabetes in humans. Recent studies in NOD mice have shown that this disease is a result of epistatic interactions between multiple genes, both inside and outside the major histocompatibility complex (MHC), generating T cells reactive against an expanding repertoire of autoantigens.

title: Expression of an activating mutation in the gene encoding the KATP channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes.
abstract: Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K+ (KATP) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of KATP channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of KATP channels, closed KATP channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of KATP channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.

title: Cardiac Dysfunction and Metabolic Inflexibility in a Mouse Model of Diabetes Without Dyslipidemia
abstract: Diabetes is a well-established risk factor for heart disease, leading to impaired cardiac function and a metabolic switch toward fatty acid usage. In this study, we investigated if hyperglycemia/hypoinsulinemia in the absence of dyslipidemia is sufficient to drive these changes and if they can be reversed by restoring euglycemia. Using the βV59M mouse model, in which diabetes can be rapidly induced and reversed, we show that stroke volume and cardiac output were reduced within 2 weeks of diabetes induction. Flux through pyruvate dehydrogenase was decreased, as measured in vivo by hyperpolarized [1-13C]pyruvate MRS. Metabolomics showed accumulation of pyruvate, lactate, alanine, tricarboxyclic acid cycle metabolites, and branched-chain amino acids. Myristic and palmitoleic acid were decreased. Proteomics revealed proteins involved in fatty acid metabolism were increased, whereas those involved in glucose metabolism decreased. Western blotting showed enhanced pyruvate dehydrogenase kinase 4 (PDK4) and uncoupling protein 3 (UCP3) expression. Elevated PDK4 and UCP3 and reduced pyruvate usage were present 24 h after diabetes induction. The observed effects were independent of dyslipidemia, as mice showed no evidence of elevated serum triglycerides or lipid accumulation in peripheral organs (including the heart). The effects of diabetes were reversible, as glibenclamide therapy restored euglycemia, cardiac metabolism and function, and PDK4/UCP3 levels.

title: Mice deficient in mitochondrial glycerol-3-phosphate acyltransferase-1 have diminished myocardial triacylglycerol accumulation during lipogenic diet and altered phospholipid fatty acid composition.
abstract: Glycerol-3-phosphate acyltransferase-1 (GPAT1), which is located on the outer mitochondrial membrane comprises up to 30% of total GPAT activity in the heart. It is one of at least four mammalian GPAT isoforms known to catalyze the initial, committed, and rate-limiting step of glycerolipid synthesis. Because excess triacylglycerol (TAG) accumulates in cardiomyocytes in obesity and type 2 diabetes, we determined whether lack of GPAT1 would alter the synthesis of heart TAG and phospholipids after a 2-week high-sucrose diet or a 3-month high-fat diet. Even in the absence of hypertriglyceridemia, TAG increased 2-fold with both diets in hearts from wildtype mice. In contrast, hearts from Gpat1(-/-) mice contained 20-80% less TAG than the wildtype controls. In addition, hearts from Gpat1(-/-) mice fed the high-sucrose diet incorporate 60% less [(14)C]palmitate into heart TAG as compared to wildtype mice. Because GPAT1 prefers 16:0-CoA to other long-chain acyl-CoA substrates, we determined the fatty acid composition of heart phospholipids. Compared to wildtype littermate controls, hearts from Gpat1(-/-)(-/-) mice contained a lower amount of 16:0 in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine/phosphatidylinositol and significantly more C20:4n6. Phosphatidylcholine and phosphatidylethanolamine from Gpat1(-/-)(-/-) hearts also contained higher amounts of 18:0 and 18:1. Although at least three other GPAT isoforms are expressed in the heart, our data suggest that GPAT1 contributes significantly to cardiomyocyte TAG synthesis during lipogenic or high-fat diets and influences the incorporation of 20:4n6 into heart phospholipids.

title: Studies of streptozotocin-induced insulitis and diabetes.
abstract: Multiple small injections of streptozotocin produce a delayed, progressive increase in plasma glucose in mice within 5-6 days after the injections, in association with pronounced insulitis and induction of type C viruses within beta cells. Multiple subdiabetogenic doses of streptozotocin in rats and multiple injections of another beta cell toxin, alloxan, in mice did not induce insulitis although hyperglycemia followed the injection of larger quantities of both agents. In mice, the prior injection of 3-O-methyl-D-glucose (3-OMG) or nicotinamide attenuated the diabetic syndrome produced by streptozotocin; however, 3-OMG was more protective. Rabbit antimouse lymphocyte serum, alone, provided partial protection but, when given together with either 3-OMG or nicotinamide, effectively prevented the streptozotocin-induced diabetic syndrome. Cessation of these preventive treatments was followed by the appearance of insulitis and diabetes. These findings suggest that multiple injections of streptozotocin induce, in susceptible hosts, the triad of direct beta cell cytotoxicity, virus induction within beta cells, and cell-mediated autoimmune reaction. These factors, acting separately or in concert, appear to induce a destructive insulitis and severe diabetes. The relative importance of each component and the factors governing host susceptibility remain to be clarified.

title: High‐fat Diets: Modeling the Metabolic Disorders of Human Obesity in Rodents
abstract: diet feeding can induce obesity and metabolic disorders in rodents that resemble the human metabolic syndrome. However, this dietary intervention is not standardized, and the HF‐induced phenotype varies distinctly among different studies. The question which HF diet type is best to model the metabolic deterioration seen in human obesity remains unclear. Therefore, in this review, metabolic data obtained with different HF diet approaches are compiled. Both whole‐body and organ‐specific diet effects are analyzed.

title: Diet-Induced Type II Diabetes in C57BL/6J Mice
abstract: We investigated the effects of diet-induced obesity on glucose metabolism in two strains of mice, C57BL/6J and A/J. Twenty animals from each strain received ad libitum exposure to a high–fat high-simple-carbohydrate diet or standard Purina Rodent Chow for 6 mo. Exposure to the high-fat, high-simple-carbohydrate, low-fiber diet produced obesity in both A/J and C57BL/6J mice. Whereas obesity was associated with only moderate glucose intolerance and insulin resistance in A/J mice, obese C57BL/6J mice showed clear-cut diabetes with fasting blood glucose levels of >240 mg/dl and blood insulin levels of >150 μU/ml. C57BL/6J mice showed larger glycemic responses to stress and epinephrine in the lean state than AJ mice, and these responses were exaggerated by obesity. These data suggest that the C57BL/6J mouse carries a genetic predisposition to develop non-insulin-dependent (type II) diabetes. Futhermore, altered glycemic response to adrenergic stimulation may be a biologic marker for this genetic predisposition to develop type II diabetes.

title: Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening.
abstract: The objective of the present study was to develop a rat model that replicates the natural history and metabolic characteristics of human type 2 diabetes and is also suitable for pharmacological screening. Male Sprague-Dawley rats (160-180 g) were divided into two groups and fed with commercially available normal pellet diet (NPD) (12% calories as fat) or in-house prepared high-fat diet (HFD) (58% calories as fat), respectively, for a period of 2 weeks. The HFD-fed rats exhibited significant increase in body weight, basal plasma glucose (PGL), insulin (PI), triglycerides (PTG) and total cholesterol (PTC) levels as compared to NPD-fed control rats. Besides, the HFD rats showed significant reduction in glucose disappearance rate (K-value) on intravenous insulin glucose tolerance test (IVIGTT). Hyperinsulinemia together with reduced glucose disappearance rate (K-value) suggested that the feeding of HFD-induced insulin resistance in rats. After 2 weeks of dietary manipulation, a subset of the rats from both groups was injected intraperitoneally with low dose of streptozotocin (STZ) (35 mg kg(-1)). Insulin-resistant HFD-fed rats developed frank hyperglycemia upon STZ injection that, however, caused only mild elevation in PGL in NPD-fed rats. Though there was significant reduction in PI level after STZ injection in HFD rats, the reduction observed was only to a level that was comparable with NPD-fed control rats. In addition, the levels of PTG and PTC were further accentuated after STZ treatment in HFD-fed rats. In contrast, STZ (35 mg kg(-1), i.p.) failed to significantly alter PI, PTG and PTC levels in NPD-fed rats. Thus, these fat-fed/STZ-treated rats simulate natural disease progression and metabolic characteristics typical of individuals at increased risk of developing type 2 diabetes because of insulin resistance and obesity. Further, the fat-fed/STZ-treated rats were found to be sensitive for glucose lowering effects of insulin sensitizing (pioglitazone) as well as insulinotropic (glipizide) agents. Besides, the effect of pioglitazone and glipizide on the plasma lipid parameters (PTG and PTC) was shown in these diabetic rats. The present study represents that the combination of HFD-fed and low-dose STZ-treated rat serves as an alternative animal model for type 2 diabetes simulating the human syndrome that is also suitable for testing anti-diabetic agents for the treatment of type 2 diabetes.

title: Cardiac metabolism in a new rat model of type 2 diabetes using high-fat diet with low dose streptozotocin
abstract: BackgroundTo study the pathogenesis of diabetic cardiomyopathy, reliable animal models of type 2 diabetes are required. Physiologically relevant rodent models are needed, which not only replicate the human pathology but also mimic the disease process. Here we characterised cardiac metabolic abnormalities, and investigated the optimal experimental approach for inducing disease, in a new model of type 2 diabetes. Wistar rats were fed a high-fat diet for three weeks, with a single intraperitoneal injection of low dose streptozotocin (STZ) after fourteen days at 15, 20, 25 or 30 mg/kg body weight. Compared with chow-fed or high-fat diet fed control rats, a high-fat diet in combination with doses of 15–25 mg/kg STZ did not change insulin concentrations and rats maintained body weight. In contrast, 30 mg/kg STZ induced hypoinsulinaemia, hyperketonaemia and weight loss. There was a dose-dependent increase in blood glucose and plasma lipids with increasing concentrations of STZ. Cardiac and hepatic triglycerides were increased by all doses of STZ, in contrast, cardiac glycogen concentrations increased in a dose-dependent manner with increasing STZ concentrations. Cardiac glucose transporter 4 protein levels were decreased, whereas fatty acid metabolism-regulated proteins, including uncoupling protein 3 and pyruvate dehydrogenase (PDH) kinase 4, were increased with increasing doses of STZ. Cardiac PDH activity displayed a dose-dependent relationship between enzyme activity and STZ concentration. Cardiac insulin-stimulated glycolytic rates were decreased by 17% in 15 mg/kg STZ high-fat fed diabetic rats compared with control rats, with no effect on cardiac contractile function.ConclusionsHigh-fat feeding in combination with a low dose of STZ induced cardiac metabolic changes that mirror the decrease in glucose metabolism and increase in fat metabolism in diabetic patients. While low doses of 15–25 mg/kg STZ induced a type 2 diabetic phenotype, higher doses more closely recapitulated type 1 diabetes, demonstrating that the severity of diabetes can be modified according to the requirements of the study.

title: Cardiac diastolic dysfunction in high-fat diet fed mice is associated with lipotoxicity without impairment of cardiac energetics in vivo.
abstract: Obesity is often associated with abnormalities in cardiac morphology and function. This study tested the hypothesis that obesity-related cardiomyopathy is caused by impaired cardiac energetics. In a mouse model of high-fat diet (HFD)-induced obesity, we applied in vivo cardiac (31)P magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) to investigate cardiac energy status and function, respectively. The measurements were complemented by ex vivo determination of oxygen consumption in isolated cardiac mitochondria, the expression of proteins involved in energy metabolism, and markers of oxidative stress and calcium homeostasis. We also assessed whether HFD induced myocardial lipid accumulation using in vivo (1)H MRS, and if this was associated with apoptosis and fibrosis. Twenty weeks of HFD feeding resulted in early stage cardiomyopathy, as indicated by diastolic dysfunction and increased left ventricular mass, without any effects on systolic function. In vivo cardiac phosphocreatine-to-ATP ratio and ex vivo oxygen consumption in isolated cardiac mitochondria were not reduced after HFD feeding, suggesting that the diastolic dysfunction was not caused by impaired cardiac energetics. HFD feeding promoted mitochondrial adaptations for increased utilization of fatty acids, which was however not sufficient to prevent the accumulation of myocardial lipids and lipid intermediates. Myocardial lipid accumulation was associated with oxidative stress and fibrosis, but not apoptosis. Furthermore, HFD feeding strongly reduced the phosphorylation of phospholamban, a prominent regulator of cardiac calcium homeostasis and contractility. In conclusion, HFD-induced early stage cardiomyopathy in mice is associated with lipotoxicity-associated oxidative stress, fibrosis, and disturbed calcium homeostasis, rather than impaired cardiac energetics.

title: Increasing Pyruvate Dehydrogenase Flux as a Treatment for Diabetic Cardiomyopathy: A Combined 13C Hyperpolarized Magnetic Resonance and Echocardiography Study
abstract: Although diabetic cardiomyopathy is widely recognized, there are no specific treatments available. Altered myocardial substrate selection has emerged as a candidate mechanism behind the development of cardiac dysfunction in diabetes. As pyruvate dehydrogenase (PDH) activity appears central to the balance of substrate use, we aimed to investigate the relationship between PDH flux and myocardial function in a rodent model of type 2 diabetes and to explore whether or not increasing PDH flux, with dichloroacetate, would restore the balance of substrate use and improve cardiac function. All animals underwent in vivo hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopy and echocardiography to assess cardiac PDH flux and function, respectively. Diabetic animals showed significantly higher blood glucose levels (10.8 ± 0.7 vs. 8.4 ± 0.5 mmol/L), lower PDH flux (0.005 ± 0.001 vs. 0.017 ± 0.002 s-1), and significantly impaired diastolic function (transmitral early diastolic peak velocity/early diastolic myocardial velocity ratio [ 12.2 ± 0.8 vs. 20 ± 2), which are in keeping with early diabetic cardiomyopathy. Twenty-eight days of treatment with dichloroacetate restored PDH flux to normal levels (0.018 ± 0.002 s-1), reversed diastolic dysfunction (E/E′ 14 ± 1), and normalized blood glucose levels (7.5 ± 0.7 mmol/L). The treatment of diabetes with dichloroacetate therefore restored the balance of myocardial substrate selection, reversed diastolic dysfunction, and normalized blood glucose levels. This suggests that PDH modulation could be a novel therapy for the treatment and/or prevention of diabetic cardiomyopathy.

title: Effect of a high fat diet on the metabolism and mechanical behaviour of the isolated rat heart.
abstract: The effects of a high fat, atherogenic diet was studied on the metabolism and mechanical behaviour of the isolated perfused rat heart. Palmitate and pyruvate metabolism were similar in control and fat-fed perfused hearts. Glucose uptake was lower in the fat-fed perfused heart, but the percentages of glucose converted to 14CO2 and glycogen were similar for the two groups. Explanations for the differences in glucose uptake are discussed. Fat-fed perfused hearts, with glucose as substrate, had significantly lower values for spontaneous rate, stress relaxation and tension time per minute than control hearts. With palmitate as substrate, the fat-fed perfused hearts had decreased coronary flow rates and a greater rate of tension increase on contraction. Contraction characteristics were different when hearts were perfused with glucose, compared with palmitate, in both dietary groups.

title: A high fat diet increases mitochondrial fatty acid oxidation and uncoupling to decrease efficiency in rat heart
abstract: Elevated levels of cardiac mitochondrial uncoupling protein 3 (UCP3) and decreased cardiac efficiency (hydraulic power/oxygen consumption) with abnormal cardiac function occur in obese, diabetic mice. To determine whether cardiac mitochondrial uncoupling occurs in non-genetic obesity, we fed rats a high fat diet (55% kcal from fat) or standard laboratory chow (7% kcal from fat) for 3 weeks, after which we measured cardiac function in vivo using cine MRI, efficiency in isolated working hearts and respiration rates and ADP/O ratios in isolated interfibrillar mitochondria; also, measured were medium chain acyl-CoA dehydrogenase (MCAD) and citrate synthase activities plus uncoupling protein 3 (UCP3), mitochondrial thioesterase 1 (MTE-1), adenine nucleotide translocase (ANT) and ATP synthase protein levels. We found that in vivo cardiac function was the same for all rats, yet oxygen consumption was 19% higher in high fat-fed rat hearts, therefore, efficiency was 21% lower than in controls. We found that mitochondrial fatty acid oxidation rates were 25% higher, and MCAD activity was 23% higher, in hearts from rats fed the high fat diet when compared with controls. Mitochondria from high fat-fed rat hearts had lower ADP/O ratios than controls, indicating increased respiratory uncoupling, which was ameliorated by GDP, a UCP3 inhibitor. Mitochondrial UCP3 and MTE-1 levels were both increased by 20% in high fat-fed rat hearts when compared with controls, with no significant change in ATP synthase or ANT levels, or citrate synthase activity. We conclude that increased cardiac oxygen utilisation, and thereby decreased cardiac efficiency, occurs in non-genetic obesity, which is associated with increased mitochondrial uncoupling due to elevated UCP3 and MTE-1 levels.

title: Exercise of obese mice induces cardioprotection and oxygen sparing in hearts exposed to high-fat load.
abstract: Exercise training is a potent therapeutic approach in obesity and diabetes that exerts protective effects against the development of diabetic cardiomyopathy and ischemic injury. Acute increases in circulating fatty acids (FAs) during an ischemic insult can challenge the heart, since high FA load is considered to have adverse cardiac effects. In the present study, we tested the hypothesis that exercise-induced cardiac effects in diet-induced obese mice are abrogated by an acute high FA load. Diet-induced obese mice were fed a high-fat diet (HFD) for 20 wk. They were exercised using moderate- and/or high-intensity exercise training (MIT and HIT, respectively) for 10 or 3 wk, and isolated perfused hearts from these mice were exposed to a high FA load. Sedentary HFD mice served as controls. Ventricular function and myocardial O2 consumption were assessed after 10 wk of HIT and MIT, and postischemic functional recovery and infarct size were examined after 3 wk of HIT. In addition to improving aerobic capacity and reducing obesity and insulin resistance, long-term exercise ameliorated the development of diet-induced cardiac dysfunction. This was associated with improved mechanical efficiency because of reduced myocardial oxygen consumption. Although to a lesser extent, 3-wk HIT also increased aerobic capacity and decreased obesity and insulin resistance. HIT also improved postischemic functional recovery and reduced infarct size. Event upon the exposure to a high FA load, short-term exercise induced an oxygen-sparing effect. This study therefore shows that exercise-induced cardioprotective effects are present under hyperlipidemic conditions and highlights the important role of myocardial energetics during ischemic stress.NEW & NOTEWORTHY The exercise-induced cardioprotective effects in obese hearts are present under hyperlipidemic conditions, comparable to circulating levels of FA occurring with an ischemic insult. Myocardial oxygen sparing is associated with this effect, despite the general notion that high fat can decrease cardiac efficiency. This highlights the role of myocardial energetics during ischemic stress.

title: THE CHEMICAL NATURE OF HEART FAILURE
abstract: Excerpt Heart failure in its commonest and simplest form represents an inadequacy in contraction of a critical number of individual heart muscle cells. Every clinician has witnessed the death of pa...

title: The creatine kinase system and pleiotropic effects of creatine
abstract: The pleiotropic effects of creatine (Cr) are based mostly on the functions of the enzyme creatine kinase (CK) and its high-energy product phosphocreatine (PCr). Multidisciplinary studies have established molecular, cellular, organ and somatic functions of the CK/PCr system, in particular for cells and tissues with high and intermittent energy fluctuations. These studies include tissue-specific expression and subcellular localization of CK isoforms, high-resolution molecular structures and structure–function relationships, transgenic CK abrogation and reverse genetic approaches. Three energy-related physiological principles emerge, namely that the CK/PCr systems functions as (a) an immediately available temporal energy buffer, (b) a spatial energy buffer or intracellular energy transport system (the CK/PCr energy shuttle or circuit) and (c) a metabolic regulator. The CK/PCr energy shuttle connects sites of ATP production (glycolysis and mitochondrial oxidative phosphorylation) with subcellular sites of ATP utilization (ATPases). Thus, diffusion limitations of ADP and ATP are overcome by PCr/Cr shuttling, as most clearly seen in polar cells such as spermatozoa, retina photoreceptor cells and sensory hair bundles of the inner ear. The CK/PCr system relies on the close exchange of substrates and products between CK isoforms and ATP-generating or -consuming processes. Mitochondrial CK in the mitochondrial outer compartment, for example, is tightly coupled to ATP export via adenine nucleotide transporter or carrier (ANT) and thus ATP-synthesis and respiratory chain activity, releasing PCr into the cytosol. This coupling also reduces formation of reactive oxygen species (ROS) and inhibits mitochondrial permeability transition, an early event in apoptosis. Cr itself may also act as a direct and/or indirect anti-oxidant, while PCr can interact with and protect cellular membranes. Collectively, these factors may well explain the beneficial effects of Cr supplementation. The stimulating effects of Cr for muscle and bone growth and maintenance, and especially in neuroprotection, are now recognized and the first clinical studies are underway. Novel socio-economically relevant applications of Cr supplementation are emerging, e.g. for senior people, intensive care units and dialysis patients, who are notoriously Cr-depleted. Also, Cr will likely be beneficial for the healthy development of premature infants, who after separation from the placenta depend on external Cr. Cr supplementation of pregnant and lactating women, as well as of babies and infants are likely to be of benefit for child development. Last but not least, Cr harbours a global ecological potential as an additive for animal feed, replacing meat- and fish meal for animal (poultry and swine) and fish aqua farming. This may help to alleviate human starvation and at the same time prevent over-fishing of oceans.

title: Diastolic dysfunction is associated with altered myocardial metabolism in asymptomatic normotensive patients with well-controlled type 2 diabetes mellitus.
abstract: This study evaluated myocardial function in relation to high-energy phosphate (HEP) metabolism in asymptomatic patients with uncomplicated type 2 diabetes mellitus using magnetic resonance (MR) techniques.



Myocardial dysfunction may occur in patients with type 2 diabetes mellitus in the absence of coronary artery disease or left ventricular (LV) hypertrophy. The mechanisms underlying this diabetic cardiomyopathy are largely unknown, but may involve altered myocardial energy metabolism.



We assessed myocardial systolic and diastolic function and HEP metabolism in 12 asymptomatic normotensive male patients with recently diagnosed, well-controlled type 2 diabetes and 12 controls, using MR imaging and phosphorus-31-nuclear MR spectroscopy (31P-MRS) on a 1.5 T clinical scanner; 31P-MR spectra were quantified, and myocardial HEP metabolism was expressed as phosphocreatine to adenosine-triphosphate (PCr/ATP) ratio.



No differences were found in LV mass and systolic function between patients and controls. However, early (E) acceleration peak, deceleration peak, peak filling rate, and transmitral early-to-late diastolic peak flow (E/A) ratio, all indexes of diastolic function, were significantly decreased in patients compared with controls (p < 0.02). In addition, myocardial PCr/ATP in patients was significantly lower than in controls (1.47 vs. 1.88, p < 0.01). Inverse associations were found between myocardial PCr/ATP and E acceleration peak, E deceleration peak, and E peak filling rate (all, p < 0.05).



These results indicate that altered myocardial energy metabolism may contribute to LV diastolic functional changes in patients with recently diagnosed, well-controlled and uncomplicated type 2 diabetes.

title: Abnormal Cardiac and Skeletal Muscle Energy Metabolism in Patients With Type 2 Diabetes
abstract: It is well known that patients with type 2 diabetes have increased risk of cardiovascular disease, but it is not known whether they have underlying abnormalities in cardiac or skeletal muscle high‐energy phosphate metabolism.  We studied 21 patients with type 2 diabetes with no evidence of coronary artery disease or impaired cardiac function, as determined by echocardiography, and 15 age‐, sex‐, and body mass index‐matched control subjects. Cardiac high‐energy phosphate metabolites were measured at rest using 31P nuclear magnetic resonance spectroscopy (MRS). Skeletal muscle high‐energy phosphate metabolites, intracellular pH, and oxygenation were measured using 31P MRS and near infrared spectrophotometry, respectively, before, during, and after exercise. Although their cardiac morphology, mass, and function appeared to be normal, the patients with diabetes had significantly lower phosphocreatine (PCr)/ATP ratios, at 1.50±0.11, than the healthy volunteers, at 2.30±0.12. The cardiac PCr/ATP ratios correlated negatively with the fasting plasma free fatty acid concentrations. Although skeletal muscle energetics and pH were normal at rest, PCr loss and pH decrease were significantly faster during exercise in the patients with diabetes, who had lower exercise tolerance. After exercise, PCr recovery was slower in the patients with diabetes and correlated with tissue reoxygenation times. The exercise times correlated negatively with the deoxygenation rates and the hemoglobin (Hb)A1c levels and the reoxygenation times correlated positively with the HbA1c levels.  Type 2 diabetic patients with apparently normal cardiac function have impaired myocardial and skeletal muscle energy metabolism related to changes in circulating metabolic substrates.

title: Diabetic db/db mice do not develop heart failure upon pressure overload: a longitudinal in vivo PET, MRI, and MRS study on cardiac metabolic, structural, and functional adaptations
abstract: Heart failure is associated with altered myocardial substrate metabolism and impaired cardiac energetics. Comorbidities like diabetes may influence the metabolic adaptations during heart failure development. We quantified to what extent changes in substrate preference, lipid accumulation, and energy status predict the longitudinal development of hypertrophy and failure in the non-diabetic and the diabetic heart.



Transverse aortic constriction (TAC) was performed in non-diabetic (db/+) and diabetic (db/db) mice to induce pressure overload. Magnetic resonance imaging, 31P magnetic resonance spectroscopy (MRS), 1H MRS, and 18F-fluorodeoxyglucose-positron emission tomography (PET) were applied to measure cardiac function, energy status, lipid content, and glucose uptake, respectively. In vivo measurements were complemented with ex vivo techniques of high-resolution respirometry, proteomics, and western blotting to elucidate the underlying molecular pathways. In non-diabetic mice, TAC induced progressive cardiac hypertrophy and dysfunction, which correlated with increased protein kinase D-1 (PKD1) phosphorylation and increased glucose uptake. These changes in glucose utilization preceded a reduction in cardiac energy status. At baseline, compared with non-diabetic mice, diabetic mice showed normal cardiac function, higher lipid content and mitochondrial capacity for fatty acid oxidation, and lower PKD1 phosphorylation, glucose uptake, and energetics. Interestingly, TAC affected cardiac function only mildly in diabetic mice, which was accompanied by normalization of phosphorylated PKD1, glucose uptake, and cardiac energy status.



The cardiac metabolic adaptations in diabetic mice seem to prevent the heart from failing upon pressure overload, suggesting that restoring the balance between glucose and fatty acid utilization is beneficial for cardiac function.

title: Cardiac remodeling rather than disturbed myocardial energy metabolism is associated with cardiac dysfunction in diabetic rats.
abstract: Diabetes mellitus (DM) alters the energy substrate metabolism in the heart and the early sign of diabetic cardiomyopathy is the diastolic dysfunction. Although it is known that the extracellular matrix must be altered in the presence of diabetes, its local regulation has not been fully elucidated. Our aim was to evaluate in vivo left ventricular (LV) structure; function and bioenergetics in streptozotocin (STZ) induced diabetes mellitus.



Cardiac function was evaluated using echocardiography in anesthetized Sprague–Dawley rats 12 weeks after injection of STZ and in age-matched control rats before and after atrial pacing. In vivo ³¹P magnetic resonance spectroscopy was done to measure the phosphocreatine (PCr) to ATP ratio. Myocardial protein expression of metalloproteinases MMP-2, -9, tissue inhibitor TIMP-1, -2 and collagen was measured using Western blot.



Bodyweight (BW) was decreased in diabetic rats. Heart weight/BW and LV mass/BW ratios were higher in diabetic animals compared to controls (2.3 ± 08 vs 2.1 ± 08 mg/g p <0.05). Heart rate was lower in diabetic rats (293 ± 20 vs 394 ± 36 bpm p <0.05). The velocity of circumferential shortening and peak aortic velocity were lower in diabetic animals and were more pronounced during atrial pacing. The basal PCr/ATP ratio was not different in the two groups. Total collagen was higher in diabetic rats (3.8 ± 0.3 vs 2.9 ± 01 mg/g, p <0.05). Protein expression of MMP-2 was significantly diminished in diabetic rats by ≈ 60%, while MMP-9, TIMP-1 and -2 were unchanged.



Streptozotocin induced diabetes led to increased LV/bodyweight, increased collagen content, and diminished MMP-2 with no change in PCr/ATP. Therefore, remodeling rather than disturbed energetics may underlie diabetic cardiomyopathy.

title: Single dose streptozotocin-induced diabetes: considerations for study design in islet transplantation models
abstract: Streptozotocin (STZ)-induced diabetes mellitus (DM) offers a very cost-effective and expeditious technique that can be used in most strains of rodents, opening the field of DM research to an array of genotypic and phenotypic options that would otherwise be inaccessible. Despite widespread use of STZ in small animal models, the data available concerning drug preparation, dosing and administration, time to onset and severity of DM, and any resulting moribundity and mortality are often limited and inconsistent. Because of this, investigators inexperienced with STZ-induced diabetes may find it difficult to precisely design new studies with this potentially toxic chemical and account for the severity of DM it is capable of inducing. Until a better option becomes available, attempts need to be made to address shortcomings with current STZ-induced DM models. In this paper we review the literature and provide data from our pancreatic islet transplantation experiments using single high-dose STZ-induced DM in NCr athymic nude mice with hopes of providing clarification for study design, suggesting refinements to the process, and developing a more humane process of chemical diabetes induction.

title: A high-fat diet impairs cardiac high-energy phosphate metabolism and cognitive function in healthy human subjects.
abstract: High-fat, low-carbohydrate diets are widely used for weight reduction, but they may also have detrimental effects via increased circulating free fatty acid concentrations.



We tested whether raising plasma free fatty acids by using a high-fat, low-carbohydrate diet results in alterations in heart and brain in healthy subjects.



Men (n = 16) aged 22 ± 1 y (mean ± SE) were randomly assigned to 5 d of a high-fat, low-carbohydrate diet containing 75 ± 1% of calorie intake through fat consumption or to an isocaloric standard diet providing 23 ± 1% of calorie intake as fat. In a crossover design, subjects undertook the alternate diet after a 2-wk washout period, with results compared after the diet periods. Cardiac (31)P magnetic resonance (MR) spectroscopy and MR imaging, echocardiography, and computerized cognitive tests were used to assess cardiac phosphocreatine (PCr)/ATP, cardiac function, and cognitive function, respectively.



Compared with the standard diet, subjects who consumed the high-fat, low-carbohydrate diet had 44% higher plasma free fatty acids (P < 0.05), 9% lower cardiac PCr/ATP (P < 0.01), and no change in cardiac function. Cognitive tests showed impaired attention (P < 0.01), speed (P < 0.001), and mood (P < 0.01) after the high-fat, low-carbohydrate diet.



Raising plasma free fatty acids decreased myocardial PCr/ATP and reduced cognition, which suggests that a high-fat diet is detrimental to heart and brain in healthy subjects.

title: Effects of weight loss on myocardial energetics and diastolic function in obesity
abstract: A reduced myocardial phosphocreatine/adenosine triphosphate (PCr/ATP) ratio is linked to both diastolic dysfunction and heart failure. Although obesity is well known to cause diastolic dysfunction a link to impaired cardiac energetics has only recently been established. We assessed whether or not long-term weight loss in obesity, which is known to reduce mortality, is accompanied by both improved cardiac energetics and diastolic function. Normal weight (BMI 22 ± 2; n = 18) and obese subjects (BMI 34 ± 4; n = 13) underwent cine-MRI (1.5 Tesla) to determine left ventricular diastolic function using volume-time curve analysis, and 31P-MR spectroscopy (3 Tesla) to assess cardiac energetics (PCr/ATP ratio). Obese subjects (n = 13) underwent repeat assessment after 1 year of supervised weight loss. Obesity, in the absence of identifiable cardiovascular risk factors, was associated with significantly impaired myocardial high energy phosphate metabolism (PCr/ATP ratio, normal; 2.03 ± 0.27 vs. obese; 1.58 ± 0.47, p = 0.002) and significantly lower peak diastolic filling rate (normal; 4.8 ± 0.8 vs. obese; 3.8 ± 0.7 EDV/s, p = 0.01). Weight loss (on average 9 kg, 55 % excess weight) over 1 year resulted in a 24 % increase in PCr/ATP ratio (p = 0.01) and an 18 % improvement in peak diastolic filling rate (p = 0.01). Myocardial PCr/ATP ratio remained positively correlated with peak diastolic filling rate after weight loss (r = 0.63, p = 0.02). In obesity, weight loss improves impaired cardiac energetics and myocardial relaxation. Improved myocardial energetics appear to play a key role in diastolic functional recovery accompanying weight loss.

title: Relationship between cardiac function and substrate oxidation in hearts of diabetic rats.
abstract: The effects of streptozotocin-induced diabetes on myocardial substrate oxidation and contractile function were investigated using 13C nuclear magnetic resonance (NMR) spectroscopy. To determine the consequences of diabetes on glucose oxidation, hearts were perfused with [1-13C]glucose (11 mM) alone as well as in the presence of insulin (to stimulate glucose transport) and dichloroacetate (to stimulate pyruvate dehydrogenase). The contribution of glucose to the tricarboxylic acid (TCA) cycle was significantly decreased in hearts from diabetic animals compared with controls, with glucose alone and with insulin; however, the addition of dichloroacetate significantly increased the contribution of glucose to the TCA cycle. Contractile function in hearts from diabetic animals was significantly depressed with glucose as the sole substrate, regardless of the presence of insulin or dichloroacetate (P < 0.0005). To determine whether diabetes had any direct effects on beta-oxidation and the TCA cycle, hearts were perfused with glucose (11 mM) plus [6-13C]hexanoate (0.5 mM) as substrates. In control hearts, with glucose plus hexanoate as substrates, hexanoate contributed 98.9 +/- 2% of the substrate entering the TCA cycle; this was significantly decreased to 90.7 +/- 0.6% in the diabetic group (P < 0.02). The addition of hexanoate to the perfusate resulted in a significant increase in peak systolic pressure in the diabetic group (P < 0.001) such that contractile function was indistinguishable from controls.

title: Impact of 1 wk of diabetes on the regulation of myocardial carbohydrate and fatty acid oxidation.
abstract: 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.

title: Metabolic (In)Flexibility of the Diabetic Heart
abstract: IntroductionMetabolic inflexibility was first described as a failure of skeletal muscle of diabetic subjects to appropriately move between use of lipid in the fasting state and use of carbohydrate in the insulin-stimulated prandial state. Metabolically healthy hearts have a well developed capacity to switch between lipid and carbohydrate fuels, depending on hormone levels and substrate availability in the circulation, but it is assumed that this flexibility is lost in the maladapted diabetic heart.ObjectivesWe show in this short review that chronic treatment with lipid-lowering drugs, as well as acute administration of insulin and glucose, modulate the substrate flux in the diabetic heart. We also show that such interventions have functional implications in terms of improved cardiac efficiency and tolerance to ischemic stress.

title: Increased myocardial fatty acid metabolism in patients with type 1 diabetes mellitus.
abstract: The purpose of this study was to determine if myocardial fatty acid utilization (MFAU) and myocardial fatty acid oxidation (MFAO) are increased in diabetic patients.



Experimental models of diabetes mellitus demonstrate that MFAU and MFAO are increased, and that this dependence on myocardial fatty acid metabolism may be detrimental to cardiac function. Whether similar metabolic changes occur in humans with diabetes mellitus is unclear.



Eleven healthy non-diabetic control patients (5 women, ages 25 +/- 5 years) and 11 otherwise healthy patients with type 1 diabetes mellitus (T1DM) (8 women, ages 36 +/- 10 years, HbA1c 8.4 +/- 1.9%) underwent positron emission tomography for the determination of myocardial blood flow (MBF); myocardial oxygen consumption (MVO2); myocardial glucose utilization (MGU); and MFAU, MFAO, and %MFAO.



Plasma lactate, insulin, and MBF levels were similar between the two groups. However, plasma glucose (5.71 +/- 0.98 mumol/ml vs. 5.28 +/- 0.65 mumol/ml, p = 0.04), free fatty acid levels (0.60 +/- 0.24 mumol/ml vs. 0.19 +/- 0.07 mumol/ml, p < 0.0001), and MVO2 (6.64 +/- 2.21 vs. 4.51 +/- 1.39 mumol/g/min, p = 0.007) levels were higher in the T1DM subjects. Furthermore, compared with control patients, T1DM subjects exhibited higher MFAU (213 +/- 135 nmol/g/min vs. 57 +/- 28 nmol/g/min, p = 0.0004), MFAO (206 +/- 131 nmol/g/min s. 50 +/- 26 nmol/g/min, p = 0.0002), and %MFAO (94 +/- 6% vs. 81 +/- 19%, respectively, p = 0.04). In contrast, MGU was lower in T1DM subjects than in controls (207 +/- 108 nmol/g/min vs. 403 +/- 191 nmol/g/min, p = 0.0008).



Humans with diabetes mellitus exhibit increased MFAU and MFAO and reduced MGU consistent with observations obtained in experimental models of diabetes.

title: Increased myocardial oxygen consumption reduces cardiac efficiency in diabetic mice.
abstract: Altered cardiac metabolism and function (diabetic cardiomyopathy) has been observed in diabetes. We hypothesize that cardiac efficiency, the ratio of cardiac work (pressure-volume area [PVA]) and myocardial oxygen consumption (MVo(2)), is reduced in diabetic hearts. Experiments used ex vivo working hearts from control db/+, db/db (type 2 diabetes), and db/+ mice given streptozotocin (STZ; type 1 diabetes). PVA and ventricular function were assessed with a 1.4-F pressure-volume catheter at low (0.3 mmol/l) and high (1.4 mmol/l) fatty acid concentrations with simultaneous measurements of MVo(2). Substrate oxidation and mitochondrial respiration were measured in separate experiments. Diabetic hearts showed decreased cardiac efficiency, revealed as an 86 and 57% increase in unloaded MVo(2) in db/db and STZ-administered hearts, respectively. The slope of the PVA-MVo(2) regression line was increased for db/db hearts after elevation of fatty acids, suggesting that contractile inefficiency could also contribute to the overall reduction in cardiac efficiency. The end-diastolic and end-systolic pressure-volume relationships in db/db hearts were shifted to the left with elevated end-diastolic pressure, suggesting left ventricular remodeling and/or myocardial stiffness. Thus, by means of pressure-volume technology, we have for the first time documented decreased cardiac efficiency in diabetic hearts caused by oxygen waste for noncontractile purposes.

title: Defective Oxidative Metabolism of Heart Mitochondria from Genetically Diabetic Mice
abstract: Long chain saturated β-hydroxy fatty acid content and oxidative metabolism were studied in hearts of diabetic mice (C57BL/KsJ db/db) with a progressive cardiomy-opathy at intervals of 7, 10, 16, and 26 wk of age. Total β-hydroxy fatty acid (BHFA) content increases progressively with age in diabetic hearts with a mean value of 143.5 nmoi/g dry wt as compared with a mean of 59.6 nmol/g dry wt in control hearts. There was also a redistribution of BHFA in myocardium of diabetic mice when compared with controls, with a relative decrease in β-hydroxymyristate and an increase of β-hy-droxypalmitate. Oxidative phosphorylation studies using isolated mitochondria from diabetic mice demonstrated depressed state 3 oxidation rates with both palmityl carnitine and pyruvate as substrates. While mitochondrial NADH-oxidase activity was not statistically different from that of controls, there was a significant decrease in mitochondrial total NAD + NADH content in diabetic hearts. In addition, treatment of my-ocardfal tissue with lanthanum demonstrated an abnormal permeability of sarcolemmal, intercalated disc as well as mitochondrial membranes in myocytes of diabetic mice. The data indicate that deficiencies in total NAD + NADH content can account for the depressed state 3 oxidation of palmitylcarnitine and pyruvate in diabetic mice that in turn may explain the abnormal accumulation of BFHA. The latter could play a role in altering the permeability of cardiac cell membranes.

title: Mitochondrial complex I defect and increased fatty acid oxidation enhance protein lysine acetylation in the diabetic heart.
abstract: Cardiomyopathy is a major complication of diabetes. Our study was aimed to identify the sites of mitochondrial dysfunction and delineate its consequences on mitochondrial metabolism in a model of type 1 diabetes.



Diabetes was induced by streptozotocin injection to male Lewis rats. We found a decrease in mitochondrial biogenesis pathway and electron transport chain complex assembly that targets Complex I. Oxidation of Complex II and long-chain fatty acid substrates support the electron leak and superoxide production. Mitochondrial defects do not limit fatty acid oxidation as the heart's preferred energy source indicating that the diabetic heart has a significant reserve in Complex I- and II-supported ATP production. Both mitochondrial fatty acid oxidation and Complex I defect are responsible for increased protein lysine acetylation despite an unchanged amount of the NAD(+)-dependent mitochondrial deacetylase sirt3. We quantitatively analysed mitochondrial lysine acetylation post-translational modifications and identified that the extent of lysine acetylation on 54 sites in 22 mitochondrial proteins is higher in diabetes compared with the same sites in the control. The increased lysine acetylation of the mitochondrial trifunctional protein subunit α may be responsible for the increased fatty acid oxidation in the diabetic heart.



We identified the specific defective sites in the electron transport chain responsible for the decreased mitochondrial oxidative phosphorylation in the diabetic heart. Mitochondrial protein lysine acetylation is the common consequence of both increased fatty acid oxidation and mitochondrial Complex I defect, and may be responsible for the metabolic inflexibility of the diabetic heart.

title: Functional assessment of isolated mitochondria in vitro.
abstract: Mitochondria play a pivotal role in cellular function, not only as a major site of ATP production, but also by regulating energy expenditure, apoptosis signaling, and production of reactive oxygen species. Altered mitochondrial function is reported to be a key underlying mechanism of many pathological states and in the aging process. Functional measurements of intact mitochondria isolated from fresh tissue provides distinct information regarding the function of these organelles that complements conventional mitochondrial assays using previously frozen tissue as well as in vivo assessment using techniques such as magnetic resonance and near-infrared spectroscopy. This chapter describes the process by which mitochondria are isolated from small amounts of human skeletal muscle obtained by needle biopsy and two approaches used to assess mitochondrial oxidative capacity and other key components of mitochondrial physiology. We first describe a bioluminescent approach for measuring the rates of mitochondrial ATP production. Firefly luciferase catalyzes a light-emitting reaction whereby the substrate luciferin is oxidized in an ATP-dependent manner. A luminometer is used to quantify the light signal, which is proportional to ATP concentration. We also review a method involving polarographic measurement of oxygen consumption. Measurements of oxygen consumption, which previously required large amounts of tissue, are now feasible with very small amounts of sample obtained by needle biopsy due to recent advances in the field of high-resolution respirometry. We illustrate how careful attention to substrate combinations and inhibitors allows an abundance of unique functional information to be obtained from isolated mitochondria, including function at various energetic states, oxidative capacity with electron flow through distinct complexes, coupling of oxygen consumption to ATP production, and membrane integrity. These measurements, together with studies of mitochondrial DNA abundance, mRNA levels, protein expression, and synthesis rates of mitochondrial proteins provide insightful mechanistic information about mitochondria in a variety of tissue types.

title: Accumulation of long-chain acylcarnitine and 3-hydroxy acylcarnitine molecular species in diabetic myocardium: identification of alterations in mitochondrial fatty acid processing in diabetic myocardium by shotgun lipidomics.
abstract: Diabetic cardiomyopathy is the result of maladaptive changes in energy homeostasis. However, the biochemical mechanisms underlying dysfunctional lipid metabolism in diabetic myocardium are incompletely understood. Herein, we exploit shotgun lipidomics to demonstrate a 4-fold increase in acylcarnitines in diabetic myocardium, which was reversible upon insulin treatment. Analysis of acylcarnitine molecular species in myocardium unexpectedly identified acylcarnitine molecular species containing a mass shift of 16 amu in comparison to the anticipated molecular species. Synthesis of 3-hydroxy acylcarnitine identified the natural products as the 3-hydroxylated acylcarnitines through comparisons of diagnostic fragmentation patterns of synthetic and naturally occurring constituents using tandem mass spectrometry. Diabetes induced an increase of both calcium-independent phospholipase A(2) (iPLA(2)) mRNA and iPLA(2) activity in rat myocardium. Cardiac ischemia in myocardium genetically engineered to overexpress iPLA(2) dramatically increased the amount of acylcarnitine present in myocardium. Moreover, mechanism-based inactivation of iPLA(2) in either wild-type or transgenic myocardium ablated a substantial portion of the acylcarnitine increase. Collectively, these results identify discrete insulin remediable abnormalities in mitochondrial fatty acid processing in diabetic myocardium and identify iPLA(2) as an important enzymatic contributor to the pool of fatty acids that can be used for acylcarnitine synthesis and energy production in myocardium.

title: Acylcarnitines: reflecting or inflicting insulin resistance?
abstract: The incidence of obesity and insulin resistance is growing, and the increase in type 2 diabetes mellitus (DM2) constitutes one of the biggest challenges for our healthcare systems. Many theories are proposed for the induction of insulin resistance in glucose and lipid metabolism and its metabolic sequelae. One of these mechanisms is lipotoxicity (1–4): excess lipid supply and subsequent lipid accumulation in insulin-sensitive tissues such as skeletal muscle interfere with insulin-responsive metabolic pathways. Various lipid intermediates, like ceramides, gangliosides, diacylglycerol, and other metabolites, have been held responsible for insulin resistance (2,3,5–10). These intermediates can exert such effects because they are signaling molecules and building blocks of cellular membranes, which harbor the insulin receptor. In addition, lipids play an important role in energy homeostasis. Fatty acids (FA) can be metabolized via mitochondrial FA oxidation (FAO), which yields energy (11). As such, FAO competes with glucose oxidation in a process known as the glucose-FA, or Randle, cycle (12).

Muoio and colleagues (1,13,14) proposed an alternative mechanism in which FAO rate outpaces the tricarboxylic acid cycle (TCA), thereby leading to the accumulation of intermediary metabolites such as acylcarnitines that may interfere with insulin sensitivity. This accumulation of acylcarnitines corroborates with some human studies showing that acylcarnitines are associated with insulin resistance (15–17). In addition, acylcarnitines have a long history in the diagnosis and neonatal screening of FAO defects and other inborn errors of metabolism (18). This knowledge may aid to understand the interaction between FAO and insulin resistance and fuel future research. In this review, we discuss the role of acylcarnitines in FAO and insulin resistance as emerging from animal and human studies.

To guarantee continuous energy supply, the human body oxidizes considerable amounts of fat besides glucose. …

title: Quantitation of acyl-CoA and acylcarnitine esters accumulated during abnormal mitochondrial fatty acid oxidation.
abstract: We have used radio-high pressure liquid chromatography to study the acyl-CoA ester intermediates and the acylcarnitines formed during mitochondrial fatty acid oxidation. During oxidation of [U-14C]hexadecanoate by normal human fibroblast mitochondria, only the saturated acyl-CoA and acylcarnitine esters can be detected, supporting the concept that the acyl-CoA dehydrogenase step is rate-limiting in mitochondrial beta-oxidation. Incubations of fibroblast mitochondria from patients with defects of beta-oxidation show an entirely different profile of intermediates. Mitochondria from patients with defects in electron transfer flavoprotein and electron transfer flavoprotein:ubiquinone oxido-reductase are associated with slow flux through beta-oxidation and accumulation of long chain acyl-CoA and acylcarnitine esters. Increased amounts of saturated medium chain acyl-CoA and acylcarnitine esters are detected in the incubations of mitochondria with medium chain acyl-CoA dehydrogenase deficiency, whereas long chain 3-hydroxyacyl-CoA dehydrogenase deficiency is associated with accumulation of long chain 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters. These studies show that the control strength at the site of the defective enzyme has increased. Radio-high pressure liquid chromatography analysis of intermediates of mitochondrial fatty acid oxidation is an important new technique to study the control, organization and defects of the enzymes of beta-oxidation.

title: Type 1 diabetic cardiomyopathy in the Akita (Ins2WT/C96Y) mouse model is characterized by lipotoxicity and diastolic dysfunction with preserved systolic function.
abstract: Diabetic cardiomyopathy is an important contributor to diastolic and systolic heart failure. We examined the nature and mechanism of the cardiomyopathy in Akita (Ins2(WT/C96Y)) mice, a model of genetic nonobese type 1 diabetes that recapitulates human type 1 diabetes. Cardiac function was evaluated in male Ins2WT/C96Y and their littermate control (Ins2WT/WT) mice using echocardiography and tissue Doppler imaging, in vivo hemodynamic measurements, as well as ex vivo working heart preparation. At 3 and 6 mo of age, Ins2WT/C96Y mice exhibited preserved cardiac systolic function compared with Ins2WT/WT mice, as evaluated by ejection fraction, fractional shortening, left ventricular (LV) end-systolic pressure and maximum rate of increase in LV pressure in vivo, cardiac work, cardiac power, and rate-pressure product ex vivo. Despite the unaltered systolic function, Ins2WT/C96Y mice exhibited significant and progressive diastolic dysfunction at 3 and 6 mo of age compared with Ins2WT/WT mice as assessed by tissue and pulse Doppler imaging (E-wave velocity, isovolumetric relaxation time) and by in vivo hemodynamic measurements (LV end-diastolic pressure, time constant of LV relaxation, and maximum rate of decrease in LV pressure). We found no evidence of myocardial hypertrophy or fibrosis in the Ins2WT/C96Y myocardium. Consistent with the lack of fibrosis, expression of procollagen-alpha type I, procollagen-alpha type III, and fibronectin were not increased in these hearts. Ins2WT/C96Y hearts showed significantly reduced sarcoplasmic reticulum Ca2+-ATPase 2a (cardiac sarcoplasmic reticulum Ca2+ pump) levels, elevated beta-myosin heavy chain isoform, increased long-chain fatty acids, and triacylglycerol with evidence of lipotoxicity, as indicated by a significant rise in ceramide, diacylglycerol, and lipid deposits in the myocardium. Consistent with metabolic perturbation, and a switch to fatty acid oxidation from glucose oxidation in Ins2WT/C96Y hearts, expression of mitochondrial long-chain acyl-CoA dehydrogenase and pyruvate dehydrogenase kinase isoform 4 were increased. Insulin treatment reversed the diastolic dysfunction, the elevated B-type natriuretic peptide and beta-myosin heavy chain, and the reduced sarcoplasmic reticulum Ca2+-ATPase 2a levels with abolition of cardiac lipotoxicity. We conclude that early type 1 diabetic cardiomyopathy is characterized by diastolic dysfunction associated with lipotoxic cardiomyopathy with preserved systolic function in the absence of interstitial fibrosis and hypertrophy.

title: Lipotoxic diseases of nonadipose tissues in obesity
abstract: It is proposed that an important function of leptin is to confine the storage of triglycerides (TG) to the adipocytes, while limiting TG storage in nonadipocytes. Excess TG deposition in nonadipocytes leads to impairment of functions, increased ceramide formation, which triggers nitric oxide-mediated lipotoxicity and lipoapoptosis. The fact that TG content in nonadipocytes normally remains within a very narrow range irrespective of excess caloric intake, while TG content of adipocytes rises, is consistent with a system of fatty acid (FA) homeostasis in nonadipose tissues. When leptin is deficient or leptin receptors are dysfunctional, TG content in nonadipose tissues such as pancreatic islets, heart and skeletal muscle, can increase 10–50-fold, suggesting that leptin controls the putative homeostatic system for intracellular TG. The fact that function and viability of nonadipocytes is compromised when their TG content rises above normal implies that normal homeostasis of their intracellular FA is critical for prevention of complications of obesity. FA overload of skeletal muscle, myocardium and pancreatic islets cause, respectively, insulin resistance, lipotoxic heart disease and adipogenic type 2 diabetes. All can be completely prevented by treatment with antisteatotic agents such as troglitazone. In diet-induced obesity, leptin signaling is normal initially and lipotoxic changes are at first prevented; later, however, post-receptor leptin resistance appears, leading to dysfunction and lipoapoptosis in nonadipose tissues, the familiar complications of obesity.

title: Cardiac Steatosis in Diabetes Mellitus: A 1H-Magnetic Resonance Spectroscopy Study
abstract: The risk of heart failure in type 2 diabetes mellitus is greater than can be accounted for by hypertension and coronary artery disease. Rodent studies indicate that in obesity and type 2 diabetes mellitus, lipid overstorage in cardiac myocytes produces lipotoxic intermediates that cause apoptosis, which leads to heart failure. In humans with diabetes mellitus, cardiac steatosis previously has been demonstrated in explanted hearts of patients with end-stage nonischemic cardiomyopathy. Whether cardiac steatosis precedes the onset of cardiomyopathy in individuals with impaired glucose tolerance or in patients with type 2 diabetes mellitus is unknown.  To represent the progressive stages in the natural history of type 2 diabetes mellitus, we stratified 134 individuals (age 45±12 years) into 1 of 4 groups: (1) lean normoglycemic (lean), (2) overweight and obese normoglycemic (obese), (3) impaired glucose tolerance, and (4) type 2 diabetes mellitus. Localized 1H magnetic resonance spectroscopy and cardiac magnetic resonance imaging were used to quantify myocardial triglyceride content and left ventricular function, respectively. Compared with lean subjects, myocardial triglyceride content was 2.3-fold higher in those with impaired glucose tolerance and 2.1-fold higher in those with type 2 diabetes mellitus (P<0.05). Left ventricular ejection fraction was normal and comparable across all groups.  In humans, impaired glucose tolerance is accompanied by cardiac steatosis, which precedes the onset of type 2 diabetes mellitus and left ventricular systolic dysfunction. Thus, lipid overstorage in human cardiac myocytes is an early manifestation in the pathogenesis of type 2 diabetes mellitus and is evident in the absence of heart failure.

title: Myocardial steatosis is an independent predictor of diastolic dysfunction in type 2 diabetes mellitus.
abstract: The purpose of this study was to compare myocardial triglyceride content and function between patients with uncomplicated type 2 diabetes mellitus (T2DM) and healthy subjects within the same range of age and body mass index (BMI), and to study the associations between myocardial triglyceride content and function.



T2DM is a major risk factor for cardiovascular disease. Increasing evidence is emerging that lipid oversupply to cardiomyocytes plays a role in the development of diabetic cardiomyopathy, by causing lipotoxic injury and myocardial steatosis.



Myocardial triglyceride content and myocardial function were measured in 38 T2DM patients and 28 healthy volunteers in the same range of age and BMI by proton magnetic resonance (MR) spectroscopy and MR imaging, respectively. Myocardial triglyceride content was calculated as a percentage relative to the signal of myocardial water.



Myocardial triglyceride content was significantly higher in T2DM patients compared with healthy volunteers (0.96 +/- 0.07% vs. 0.65 +/- 0.05%, p < 0.05). Systolic function did not significantly differ between both groups. Indexes of diastolic function, including the ratio of maximal left ventricular early peak filling rate and the maximal left ventricular atrial peak filling rate (E/A) and E peak deceleration, were significantly impaired in T2DM compared with those in healthy subjects (1.08 +/- 0.04 ml/s(2) x 10(-3) vs. 1.24 +/- 0.06 ml/s(2) x 10(-3) and 3.6 +/- 0.2 ml/s(2) x 10(-3) vs. 4.4 +/- 0.3 ml/s(2) x 10(-3), respectively, p < 0.05). Multivariable analysis indicated that myocardial triglyceride content was associated with E/A and E peak deceleration, independently of diabetic state, age, BMI, heart rate, visceral fat, and diastolic blood pressure.



Myocardial triglyceride content is increased in uncomplicated T2DM and is associated with impaired left ventricular diastolic function, independently of age, BMI, heart rate, visceral fat, and diastolic blood pressure.

title: Relationship Between Left Ventricular Structural and Metabolic Remodeling in Type 2 Diabetes
abstract: Concentric left ventricular (LV) remodeling is associated with adverse cardiovascular events and is frequently observed in patients with type 2 diabetes mellitus (T2DM). Despite this, the cause of concentric remodeling in diabetes per se is unclear, but it may be related to cardiac steatosis and impaired myocardial energetics. Thus, we investigated the relationship between myocardial metabolic changes and LV remodeling in T2DM. Forty-six nonhypertensive patients with T2DM and 20 matched control subjects underwent cardiovascular magnetic resonance to assess LV remodeling (LV mass–to–LV end diastolic volume ratio), function, tissue characterization before and after contrast using T1 mapping, and 1H and 31P magnetic resonance spectroscopy for myocardial triglyceride content (MTG) and phosphocreatine-to-ATP ratio, respectively. When compared with BMI- and blood pressure–matched control subjects, subjects with diabetes were associated with concentric LV remodeling, higher MTG, impaired myocardial energetics, and impaired systolic strain indicating a subtle contractile dysfunction. Importantly, cardiac steatosis independently predicted concentric remodeling and systolic strain. Extracellular volume fraction was unchanged, indicating the absence of fibrosis. In conclusion, cardiac steatosis may contribute to concentric remodeling and contractile dysfunction of the LV in diabetes. Because cardiac steatosis is modifiable, strategies aimed at reducing MTG may be beneficial in reversing concentric remodeling and improving contractile function in the hearts of patients with diabetes.

title: Concentrations of glycerides and phospholipids in rat heart and gastrocnemius muscles. Effects of alloxan-diabetes and perfusion.
abstract: 1. Methods are described for the extraction of lipid and assay of mono-, di- and tri-glyceride glycerol and phospholipid phosphorus in rat heart and gastrocnemius muscles. 2. In hearts from normal animals, concentrations found were: monoglyceride, 0.6; diglyceride, 0.1; triglyceride, 12.6mumoles of glyceride glycerol/g. of dry muscle; phospholipid, 171mug.atoms of phospholipid phosphorus/g. of dry muscle. Concentrations of glycerides in gastrocnemius muscle were similar to heart muscle but those of phospholipids were lower (64mug.atoms of phospholipid phosphorus/g. of dry muscle). 3. Alloxan-diabetes increased the concentration of triglyceride in the muscles twofold. This increase was shown to be dependent in the heart on the availability of growth hormone and cortisol but not on the availability of dietary lipid. Total glyceride in the heart was increased after 48 and 72hr. starvation but not after 96hr. Changes in glyceride concentration seen in starvation and diabetes were not associated with significant changes in phospholipid concentration. It is suggested that mobilization of free fatty acids in diabetes leads to the synthesis of additional glyceride in muscle. 4. The possible contribution of glyceride fatty acid in the heart to respiration during perfusion has been calculated from the net loss of glyceride during perfusion, and also from the relative rates of lipolysis and esterification and compared with oxidation of fatty acid required for the balance of oxygen consumption (oxygen not utilized in the oxidation of glucose or glycogen glucose). In the normal or diabetic heart perfused with glucose and insulin the breakdown of glyceride can account for the balance of oxygen consumption. In the normal heart perfused without substrate the balance of oxygen consumption is not entirely accounted for by the breakdown of glyceride.

title: A critical role for PPARα-mediated lipotoxicity in the pathogenesis of diabetic cardiomyopathy: Modulation by dietary fat content
abstract: To explore the role of peroxisome proliferator-activated receptor α (PPARα)-mediated derangements in myocardial metabolism in the pathogenesis of diabetic cardiomyopathy, insulinopenic mice with PPARα deficiency (PPARα−/−) or cardiac-restricted overexpression [myosin heavy chain (MHC)-PPAR] were characterized. Whereas PPARα−/− mice were protected from the development of diabetes-induced cardiac hypertrophy, the combination of diabetes and the MHC-PPAR genotype resulted in a more severe cardiomyopathic phenotype than either did alone. Cardiomyopathy in diabetic MHC-PPAR mice was accompanied by myocardial long-chain triglyceride accumulation. The cardiomyopathic phenotype was exacerbated in MHC-PPAR mice fed a diet enriched in triglyceride containing long-chain fatty acid, an effect that was reversed by discontinuing the high-fat diet and absent in mice given a medium-chain triglyceride-enriched diet. Reactive oxygen intermediates were identified as candidate mediators of cardiomyopathic effects in MHC-PPAR mice. These results link dysregulation of the PPARα gene regulatory pathway to cardiac dysfunction in the diabetic and provide a rationale for serum lipid-lowering strategies in the treatment of diabetic cardiomyopathy.

title: Enhanced sarcolemmal FAT/CD36 content and triacylglycerol storage in cardiac myocytes from obese zucker rats.
abstract: In obesity, the development of cardiomyopathy is associated with the accumulation of myocardial triacylglycerols (TAGs), possibly stemming from elevation of myocardial long-chain fatty acid (LCFA) uptake. Because LCFA uptake is regulated by insulin and contractions, we examined in cardiac myocytes from lean and obese Zucker rats the effects of insulin and the contraction-mimetic agent oligomycin on the initial rate of LCFA uptake, subcellular distribution of FAT/CD36, and LCFA metabolism. In cardiac myocytes from obese Zucker rats, under basal conditions, FAT/CD36 was relocated to the sarcolemma at the expense of intracellular stores. In addition, the LCFA uptake rate, LCFA esterification rate into TAGs, and the intracellular unesterified LCFA concentration each were significantly increased. All these metabolic processes were normalized by the FAT/CD36 inhibitor sulfo-N-succinimidyloleate, indicating its antidiabetic potential. In cardiac myocytes isolated from lean rats, in vitro administration of insulin induced the translocation of FAT/CD36 to the sarcolemma and stimulated initial rates of LCFA uptake and TAG esterification. In contrast, in myocytes from obese rats, insulin failed to alter the subcellular localization of FAT/CD36 and the rates of LCFA uptake and TAG esterification. In cardiac myocytes from lean and obese animals, oligomycin stimulated the initial rates of LCFA uptake and oxidation, although oligomycin only induced the translocation of FAT/CD36 to the sarcolemma in lean rats. The present results indicate that in cardiac myocytes from obese Zucker rats, a permanent relocation of FAT/CD36 to the sarcolemma is responsible for myocardial TAG accumulation. Furthermore, in vitro these cardiac myocytes, although sensitive to contraction-like stimulation, were completely insensitive to insulin, as the basal conditions in hyperinsulinemic, obese animals resemble the insulin-stimulated condition in lean littermates.

title: Accelerated triacylglycerol turnover kinetics in hearts of diabetic rats include evidence for compartmented lipid storage.
abstract: Triacylglycerol (TAG) storage and turnover rates in the intact, beating rat heart were determined for the first time using dynamic mode (13)C- NMR spectroscopy to elucidate profound differences between hearts from diabetic rats (DR, streptozotocin treatment) and normal rats (NR). The incorporation of [2,4,6,8,10,12,14,16-(13)C(8)]palmitate into the TAG pool was monitored in isolated hearts perfused with physiological (0.5 mM palmitate, 5 mM glucose) and elevated substrate levels (1.2 mM palmitate, 11 mM glucose) characteristic of the diabetic condition. Surprisingly, although the normal hearts were enriched at a near-linear profile for >or=2 h before exponential characterization, exponential enrichment of TAG in diabetic hearts reached steady state after only 45 min. Consequently, TAG turnover rate was determined by fitting an exponential model to enrichment data rather than conventional two-point linear analysis. In the high-substrate group, both turnover rate (DR 820+/- 330, NR 190 +/-150 nmol.min(-1).g(-1) dry wt; P< 0.001) and [TAG] content (DR 78 +/-10, NR 32+/- 4 micromol/g dry wt; P< 0.001) were greater in the diabetic group. At lower substrate concentrations, turnover was greater in diabetics (DR 530+/-300, NR 160+/- 30; P<0.05). However, this could not be explained by simple mass action, because [TAG] content was similar between groups [DR 34+/- 7, NR 39+/- 9 micromol/g dry wt; not significant (NS)]. Consistent with exponential enrichment data, (13)C fractional enrichment of TAG was lower in diabetics (low- substrate groups: DR 4+/-1%, NR 10+/- 4%, P<0.05; high-substrate groups: DR 8+/- 3%, NR 14+/- 9%, NS), thereby supporting earlier speculation that TAG is compartmentalized in the diabetic heart.

title: Alterations in myocardial cardiolipin content and composition occur at the very earliest stages of diabetes: a shotgun lipidomics study.
abstract: Recently, we have identified the dramatic depletion of cardiolipin (CL) in diabetic myocardium 6 weeks after streptozotocin (STZ) injection that was accompanied by increases in triacylglycerol content and multiple changes in polar lipid molecular species. However, after 6 weeks in the diabetic state, the predominant lipid hallmarks of diabetic cardiomyopathy were each present concomitantly, and thus, it was impossible to identify the temporal course of lipid alterations in diabetic myocardium. Using the newly developed enhanced shotgun lipidomics approach, we demonstrated the dramatic loss of abundant CL molecular species in STZ-treated hearts at the very earliest stages of diabetes accompanied by a profound remodeling of the remaining CL molecular species including a 16-fold increase in the content of 18:2-22:6-22:6-22:6 CL. These alterations in CL metabolism occur within days after the induction of the diabetic state and precede the triacylglycerol accumulation manifest in diabetic myocardium. Similarly, in ob/ob mice, a dramatic and progressive redistribution from 18:2 FA-containing CL molecular species to 22:6 FA-containing CL molecular species was also identified. Collectively, these results demonstrate alterations in CL hydrolysis and remodeling at the earliest stages of diabetes and are consistent with a role for alterations in CL content in precipitating mitochondrial dysfunction in diabetic cardiomyopathy.

title: Myocardial triglyceride content and epicardial fat mass in human obesity: relationship to left ventricular function and serum free fatty acid levels.
abstract: Ectopic fat accumulation within and around the myocardial wall has been implicated in the pathogenesis of heart disease in obesity. We evaluated myocardial and epicardial fat, left ventricular (LV) function, and metabolic risk factors in nine (five lean, four moderately obese) men.



Myocardial fat percent was quantified in the septum by proton magnetic resonance spectroscopy. Reproducibility was assessed by triplicate systolic and diastolic measurements. LV parameters and epicardial fat were determined by magnetic resonance imaging. Waist-to-hip ratio and liver enzymes (alanine transaminase) were used as surrogate markers of visceral and liver fat contents.



Myocardial fat (2.1 +/- 0.5 vs. 0.8 +/- 0.1, P = 0.03) and epicardial fat (120 +/- 33 vs. 55 +/- 12 g, P = 0.08) were higher in obese than lean subjects. Individuals with above-median alanine transaminase values had a 4-fold elevation in myocardial fat. The coefficient of variation of repeated myocardial fat percent determinations was 17 +/- 3 and 23 +/- 3% in systole and diastole, respectively. Myocardial fat was correlated with free fatty acid (FFA) levels (r = 0.76; P = 0.017), epicardial fat (r = 0.69; P = 0.042), and waist-to-hip ratio (r = 0.70; P = 0.035), and it showed a tendency to associate positively with LV work. Epicardial fat was associated with peripheral vascular resistance (positively) and the cardiac index (negatively). FFA levels were significantly correlated with LV mass (r = 0.72; P = 0.030) and forward work (r = 0.74; P = 0.023).



The accumulation of triglyceride in and around the myocardium of moderately obese individuals is significant, and it is related to FFA exposure, generalized ectopic fat excess, and peripheral vascular resistance. These changes precede LV overload and hypertrophy.

title: Altered myocardial substrate metabolism is associated with myocardial dysfunction in early diabetic cardiomyopathy in rats: studies using positron emission tomography
abstract: BackgroundIn vitro data suggest that changes in myocardial substrate metabolism may contribute to impaired myocardial function in diabetic cardiomyopathy (DCM). The purpose of the present study was to study in a rat model of early DCM, in vivo changes in myocardial substrate metabolism and their association with myocardial function.MethodsZucker diabetic fatty (ZDF) and Zucker lean (ZL) rats underwent echocardiography followed by [11C]palmitate positron emission tomography (PET) under fasting, and [18F]-2-fluoro-2-deoxy-D-glucose PET under hyperinsulinaemic euglycaemic clamp conditions. Isolated cardiomyocytes were used to determine isometric force development.ResultsPET data showed a 66% decrease in insulin-mediated myocardial glucose utilisation and a 41% increase in fatty acid (FA) oxidation in ZDF vs. ZL rats (both p < 0.05). Echocardiography showed diastolic and systolic dysfunction in ZDF vs. ZL rats, which was paralleled by a significantly decreased maximal force (68%) and maximal rate of force redevelopment (69%) of single cardiomyocytes. Myocardial functional changes were significantly associated with whole-body insulin sensitivity and decreased myocardial glucose utilisation. ZDF hearts showed a 68% decrease in glucose transporter-4 mRNA expression (p < 0.05), a 22% decrease in glucose transporter-4 protein expression (p = 0.10), unchanged levels of pyruvate dehydrogenase kinase-4 protein expression, a 57% decreased phosphorylation of AMP activated protein kinase α1/2 (p < 0.05) and a 2.4-fold increased abundance of the FA transporter CD36 to the sarcolemma (p < 0.01) vs. ZL hearts, which are compatible with changes in substrate metabolism. In ZDF vs. ZL hearts a 2.4-fold reduced insulin-mediated phosphorylation of Akt was found (p < 0.05).ConclusionUsing PET and echocardiography, we found increases in myocardial FA oxidation with a concomitant decrease of insulin-mediated myocardial glucose utilisation in early DCM. In addition, the latter was associated with impaired myocardial function. These in vivo data expand previous in vitro findings showing that early alterations in myocardial substrate metabolism contribute to myocardial dysfunction.

title: Myocardial Steatosis and Biventricular Strain and Strain Rate Imaging in Patients With Type 2 Diabetes Mellitus
abstract: Magnetic resonance spectroscopy can quantify myocardial triglyceride content in type 2 diabetic patients. Its relation to alterations in left (LV) and right (RV) ventricular myocardial functions is unknown.  A total of 42 men with type 2 diabetes mellitus were recruited. Exclusion criteria included hemoglobin A1c >8.5%, known cardiovascular disease, diabetes-related complications, or blood pressure >150/85 mm Hg. Myocardial ischemia was excluded by a negative dobutamine stress test. LV and RV volumes and ejection fraction were quantified by magnetic resonance imaging. LV global longitudinal and RV free wall longitudinal strain, systolic strain rate, and diastolic strain rate were quantified by echocardiographic speckle tracking analyses. Myocardial triglyceride content was quantified by magnetic resonance spectroscopy and dichotomized on the basis of the median value of 0.76%. The median age was 59 years (25th and 75th percentiles, 54 and 62 years). Median diabetes diagnosis duration was 4 years, and median glycohemoglobin level was 6.2% (25th and 75th percentiles, 5.9% and 6.8%). There were no differences in LV and RV end-diastolic and end-systolic volume indexes and ejection fraction between patients with high (≥0.76%) and those with low (<0.76%) myocardial triglyceride content. However, patients with high myocardial triglyceride content had greater impairment of LV and RV myocardial strain and strain rate. The myocardial triglyceride content was an independent correlate of LV and RV longitudinal strain, systolic strain rate, and diastolic strain rate.  High myocardial triglyceride content is associated with more pronounced impairment of LV and RV functions in men with uncomplicated type 2 diabetes mellitus.

title: Effects of Insulin Therapy on Myocardial Lipid Content and Cardiac Geometry in Patients with Type-2 Diabetes Mellitus
abstract: Hypothesis Recent evidence suggests a link between myocardial steatosis and diabetic cardiomyopathy. Insulin, as a lipogenic and growth-promoting hormone, might stimulate intramyocardial lipid (MYCL) deposition and hypertrophy. Therefore, the aim of the present study was to investigate the short-term effects of insulin therapy (IT) on myocardial lipid content and morphology in patients with T2DM.  Eighteen patients with T2DM were recruited (age 56±2 years; HbA1c: 10.5±0.4%). In 10 patients with insufficient glucose control under oral medication IT was initiated due to secondary failure of oral glucose lowering therapy (IT-group), while 8 individuals did not require additional insulin substitution (OT-group). In order to assess MYCL and intrahepatic lipid (IHLC) content as well as cardiac geometry and function magnetic resonance spectroscopy (MRS) and imaging (MRI) examinations were performed at baseline (IT and OT) and 10 days after initiation of IT. Follow up measurements took place 181±49 days after IT.  Interestingly, basal MYCLs were 50% lower in IT- compared to OT-group (0.41±0.12 vs. 0.80±0.11% of water signal; p = 0.034). After 10 days of IT, an acute 80%-rise in MYCL (p = 0.008) was observed, while IHLC did not change. Likewise, myocardial mass (+13%; p = 0.004), wall thickness in end-diastole (+13%; p = 0.030) and concentricity, an index of cardiac remodeling, increased (+28%; p = 0.026). In the long-term MYCL returned to baseline, while IHCL significantly decreased (−31%; p = 0.000). No acute changes in systolic left ventricular function were observed.  The initiation of IT in patients with T2DM was followed by an acute rise in MYCL concentration and myocardial mass.

title: Ventricular Assist Device Implantation Corrects Myocardial Lipotoxicity, Reverses Insulin Resistance, and Normalizes Cardiac Metabolism in Patients With Advanced Heart Failure
abstract: Heart failure is associated with impaired myocardial metabolism with a shift from fatty acids to glucose use for ATP generation. We hypothesized that cardiac accumulation of toxic lipid intermediates inhibits insulin signaling in advanced heart failure and that mechanical unloading of the failing myocardium corrects impaired cardiac metabolism.  We analyzed the myocardium and serum of 61 patients with heart failure (body mass index, 26.5±5.1 kg/m2; age, 51±12 years) obtained during left ventricular assist device implantation and at explantation (mean duration, 185±156 days) and from 9 control subjects. Systemic insulin resistance in heart failure was accompanied by decreased myocardial triglyceride and overall fatty acid content but increased toxic lipid intermediates, diacylglycerol, and ceramide. Increased membrane localization of protein kinase C isoforms, inhibitors of insulin signaling, and decreased activity of insulin signaling molecules Akt and Foxo were detectable in heart failure compared with control subjects. Left ventricular assist device implantation improved whole-body insulin resistance (homeostatic model of analysis–insulin resistance, 4.5±0.6–3.2±0.5; P<0.05) and decreased myocardial levels of diacylglycerol and ceramide, whereas triglyceride and fatty acid content remained unchanged. Improved activation of the insulin/phosphatidylinositol-3 kinase/Akt signaling cascade after left ventricular assist device implantation was confirmed by increased phosphorylation of Akt and Foxo, which was accompanied by decreased membrane localization of protein kinase C isoforms after left ventricular assist device implantation.  Mechanical unloading after left ventricular assist device implantation corrects systemic and local metabolic derangements in advanced heart failure, leading to reduced myocardial levels of toxic lipid intermediates and improved cardiac insulin signaling.

title: Cardiac steatosis and left ventricular function in men with metabolic syndrome
abstract: BackgroundEctopic accumulation of fat accompanies visceral obesity with detrimental effects. Lipid oversupply to cardiomyocytes leads to cardiac steatosis, and in animal studies lipotoxicity has been associated with impaired left ventricular (LV) function. In humans, studies have yielded inconclusive results. The aim of the study was to evaluate the role of epicardial, pericardial and myocardial fat depots on LV structure and function in male subjects with metabolic syndrome (MetS).MethodsA study population of 37 men with MetS and 38 men without MetS underwent cardiovascular magnetic resonance and proton magnetic spectroscopy at 1.5 T to assess LV function, epicardial and pericardial fat area and myocardial triglyceride (TG) content.ResultsAll three fat deposits were greater in the MetS than in the control group (p <0.001). LV diastolic dysfunction was associated with MetS as measured by absolute (471 mL/s vs. 667 mL/s, p = 0.002) and normalized (3.37 s-1 vs. 3.75 s-1, p = 0.02) LV early diastolic peak filling rate and the ratio of early diastole (68% vs. 78%, p = 0.001). The amount of epicardial and pericardial fat correlated inversely with LV diastolic function. However, myocardial TG content was not independently associated with LV diastolic dysfunction.ConclusionsIn MetS, accumulation of epicardial and pericardial fat is linked to the severity of structural and functional alterations of the heart. The role of increased intramyocardial TG in MetS is more complex and merits further study.

title: Comprehensive Metabolic Profiling of Age-Related Mitochondrial Dysfunction in the High-Fat-Fed ob/ob Mouse Heart.
abstract: The ectopic deposition of fat is thought to lead to lipotoxicity and has been associated with mitochondrial dysfunction and diabetic cardiomyopathy. We have measured mitochondrial respiratory capacities in the hearts of ob/ob and wild-type mice on either a regular chow (RCD) or high-fat (HFD) diet across four age groups to investigate the impact of diet and age on mitochondrial function alongside a comprehensive strategy for metabolic profiling of the tissue. Myocardial mitochondrial dysfunction was only evident in ob/ob mice on RCD at 14 months, but it was detectable at 3 months on the HFD. Liquid chromatography-mass spectrometry (LC-MS) was used to study the profiles of acylcarnitines and the accumulation of triglycerides, but neither class of lipid was associated with mitochondrial dysfunction. However, a targeted LC-MS/MS analysis of markers of oxidative stress demonstrated increases in GSSG/GSH and 8-oxoguanine, in addition to the accumulation of diacylglycerols, which are lipid species linked to lipotoxicity. Our results demonstrate that myocardial mitochondria in ob/ob mice on RCD maintained a similar respiratory capacity to that of wild type until a late stage in aging. However, on a HFD, unlike wild-type mice, ob/ob mice failed to increase mitochondrial respiration, which may be associated with a complex I defect following increased oxidative damage.

title: Cardiac diacylglycerol accumulation in high fat-fed mice is associated with impaired insulin-stimulated glucose oxidation.
abstract: the molecular processes leading to cardiac insulin resistance induced via a high-fat diet (HFD) remain unclear. We examined the changes in cardiac insulin sensitivity and the potential mechanism(s) involved following HFD in mice.



C57BL/6 mice were fed either a low-fat diet (LFD, 4% kcal fat) or a HFD (60% kcal fat) for 3 or 10 weeks. Insulin-stimulated glucose oxidation in isolated working hearts was decreased at 10 weeks of HFD compared with mice on LFD (249 ± 19 to 399 ± 46 vs. 551 ± 97 to 1464 ± 243 nmol/g dry wt/min; P < 0.05). The accumulation of myocardial diacylglycerol (DAG; 479 ± 174 vs. 266 ± 29 micromol/g wet wt; P < 0.05), but not long-chain acyl CoA, ceramide, or triacylglycerol, correlated with the development of insulin resistance. The accumulation of DAG occurred concomitantly with an increase in glycerol phosphate acyltransferase activity, a decrease in DAG acyltransferase activity, as well as an increase in the translocation of protein kinase C-α (PKCα) and phosphorylation of p70s6k. Neither HFD-induced accumulation of cardiac DAG nor up-regulation of phosphorylated p70s6k occurred in mice lacking malonyl CoA decarboxylase which are resistant to the development of HFD-induced insulin resistance.



the activation of myocardial p70s6k and PKCα is closely associated with cardiac insulin resistance in which the accumulation of intra-myocardial DAG could be responsible.

title: Myocardium of type 2 diabetic and obese patients is characterized by alterations in sphingolipid metabolic enzymes but not by accumulation of ceramide[S]
abstract: Data from animal experiments strongly suggest that ceramide is an important mediator of lipotoxicity in the heart and that accumulation of ceramide contributes to cardiomyocyte apoptosis associated with type 2 diabetes and obesity. However, it remains unknown whether a similar relationship is present also in the human heart. Therefore, we aimed to examine whether myocardial apoptosis in obese and type 2 diabetic patients is associated with elevated ceramide level. The study included 11 lean and 26 overweight or moderately obese subjects without (n = 11, OWT) or with (n = 15, T2D-OWT) a history of type 2 diabetes. Samples of the right atrial appendage were obtained from patients at the time of coronary bypass surgery. Compared with lean subjects, the extent of DNA fragmentation (a marker of apoptosis) was significantly higher in the myocardium of OWT patients and increased further in T2D-OWT subjects. However, the content of ceramide and sphingoid bases remained stable. Interestingly, the mRNA level of enzymes involved in synthesis and degradation of ceramide including serine palmitoyltransferase, sphingosine kinase 1, neutral sphingomyelinase, and ceramidases was markedly higher in the myocardium of OWT and T2D-OWT patients compared with lean subjects. Our results indicate that in the human heart, or at least in the atrium, ceramide is not a major factor in cardiomyocyte apoptosis associated with obesity and type 2 diabetes.

title: Elevated lipid peroxidation and vitamin E-quinone levels in heart ventricles of streptozotocin-treated diabetic rats.
abstract: Diabetic patients develop cardiomyopathy characterized mainly by left ventricular contractile dysfunction and congestive heart failure. This study has investigated the effects of diabetes and insulin treatment on lipid peroxidation, vitamin E, and vitamin E-quinone levels in the heart ventricles of rat made diabetic by streptozotocin treatment. Controls were injected with buffer alone; a subgroup of diabetic rats were injected daily with insulin for 2 months. Membrane lipid peroxidation was measured by determining the thiobarbituric acid (TBA)-reactivity. Vitamin E and vitamin E-quinone were measured by using the high pressure liquid chromatography. There was a significant (p < 0.02) increase in the vitamin E-quinone in the heart ventricles of diabetic rats (0.33 +/- 0.05 microgram/mg phospholipid) compared with control rats (0.19 +/- 0.02). This increase was prevented in insulin-treated diabetic rats (0.20 +/- 0.03). Vitamin E levels were higher (14.15 +/- 1.17 micrograms/mg phospholipid) in diabetic rats compared to control rats (9.93 +/- 1.29 (p < 0.03). However, insulin treatment to diabetic rats did not cause any change in vitamin E levels (11.75 +/- 1.02) compared with diabetic rats. TBA reactivity was higher in the heart ventricles of diabetic rats (1.09 +/- 0.11 nmole/mg phospholipid) compared with controls (0.78 +/- 0.08, p < 0.04). Insulin treatment to diabetic rats prevented the increase in the lipid peroxidation (0.79 +/- 0.07); there were no statistically significant differences in TBA-reactivity levels in heart ventricles of insulin-treated diabetic and control rats. This study documents accumulation of vitamin E-quinone and lipid peroxidation products in heart ventricles in diabetic rats, which may have a role in the altered contractile property of the heart ventricles in diabetes.

title: In vivo targeted molecular magnetic resonance imaging of free radicals in diabetic cardiomyopathy within mice
abstract: Free radicals contribute to the pathogenesis of diabetic cardiomyopathy. We present a method for in vivo observation of free radical events within murine diabetic cardiomyopathy. This study reports on in vivo imaging of protein/lipid radicals using molecular MRI (mMRI) and immuno-spin trapping (IST) in diabetic cardiac muscle. To detect free radicals in diabetic cardiomyopathy, streptozotocin (STZ)-exposed mice were given 5,5-dimethyl-pyrroline-N-oxide (DMPO) and administered an anti-DMPO probe (biotin–anti-DMPO antibody–albumin–Gd–DTPA). For controls, non-diabetic mice were given DMPO (non-disease control), and administered an anti-DMPO probe; or diabetic mice were given DMPO but administered a non-specific IgG contrast agent instead of the anti-DMPO probe. DMPO administration started at 7 weeks following STZ treatment for 5 days, and the anti-DMPO probe was administered at 8 weeks for MRI detection. MRI was used to detect a significant increase (p < 0.001) in MRI signal intensity (SI) from anti-DMPO nitrone adducts in diabetic murine left-ventricular (LV) cardiac tissue, compared to controls. Regional increases in MR SI in the LV were found in the apical and upper-left areas (p < 0.01 for both), compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised cardiac tissues, which indicated elevated fluorescence only in cardiac muscle of mice administered the anti-DMPO probe. Oxidized lipids and proteins were also found to be significantly elevated (p < 0.05 for both) in diabetic cardiac muscle compared to controls. It can be concluded that diabetic mice have more heterogeneously distributed radicals in cardiac tissue than non-diabetic mice.

title: Berberine Could Ameliorate Cardiac Dysfunction via Interfering Myocardial Lipidomic Profiles in the Rat Model of Diabetic Cardiomyopathy
abstract: Diabetic cardiomyopathy (DCM) is considered to be a distinct clinical entity independent of concomitant macro- and microvascular disorders, which is initiated partly by disturbances in energy substrates. This study was to observe the dynamic modulations of berberine in DCM rats and explore the changes of lipidomic profiles of myocardial tissue.  Sprague-Dawley (SD) rats were fed high-sucrose and high-fat diet (HSHFD) for totally 22 weeks and intraperitoneally (i.p.) injected with 30 mg/kg of streptozotocin (STZ) at the fifth week to induce DCM. Seventy-two hours after STZ injection, the rats were orally given with berberine at 10, 30 mg/kg and metformin at 200 mg/kg, respectively. Dynamic changes of cardiac function, heart mass ratios and blood lipids were observed at f 4, 10, 16, and 22, respectively. Furthermore, lipid metabolites in myocardial tissue at week 16 were profiled by the ultra-high-performance liquid chromatography coupled to a quadruple time of flight mass spectrometer (UPLC/Q-TOF/MS) approach.  Berberine could protect against cardiac diastolic and systolic dysfunctions, as well as cardiac hypertrophy, and the most effective duration is with 16-week of administration. Meanwhile, 17 potential biomarkers of phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and sphingolipids (SMs) of DCM induced by HSFD/STZ were identified. The perturbations of lipidomic profiles could be partly reversed with berberine intervention, i.e., PC (16:0/20:4), PC (18:2/0:0), PC (18:0/18:2), PC (18:0/22:5), PC (20:4/0:0), PC (20:4/18:0), PC (20:4/18:1), PC (20:4/20:2), PE (18:2/0:0), and SM (d18:0/16:0).  These results indicated a close relationship between PCs, PEs and SMs and cardiac damage mechanisms during development of DCM. The therapeutic effects of berberine on DCM are partly caused by interferences with PCs, PEs, and SMs metabolisms.

title: Changes in microsomal membrane phospholipids and fatty acids and in activities of membrane-bound enzyme in diabetic rat heart
abstract: Diabetes mellitus is associated with alterations in lipid metabolism and cardiac dysfunction despite an absence of coronary arteriosclerotic changes. To investigate mechanisms of cardiac dysfunction in diabetic cardiomyopathy, we studied the relation between activities of membrane-bound enzymes and surrounding phospholipids in rats with diabetes induced with a single intravenous injection of streptozotocin (65 mg/kg). We found that total phospholipid content of sarcoplasmic reticulum membrane increased significantly 8 weeks after treatment with streptozotocin owing to increases in phosphatidylcholine and phosphatidylethanolamine, a decrease in arachidonic acid, and an increase in docosahexaenoic acid in the early stage of diabetes. Sarcolemmal Na+/K+-ATPase activity and the number of receptors decreased in isolated cardiomyoctes of diabetic rats 8 weeks after streptozotocin administration. The Ca2+ uptake of both sarcoplasmic reticulum and mitochondria decreased simultancously in permeabilized, isolated cardiomyocytes from diabetic rats. The depression of membrane-bound enzyme activities was correlated with alterations in phospholipids, which are closely related to the microenvironment of membrane-bound enzymes and influence intracellular Ca2+ metabolism. Because these changes in phospholipids and fatty acids were reversible with insulin therapy, they are diabetes-specific and might be a cause of cardiac dysfunction in diabetes.

title: A Diet Rich in Medium-Chain Fatty Acids Improves Systolic Function and Alters the Lipidomic Profile in Patients With Type 2 Diabetes: A Pilot Study.
abstract: Excessive cardiac long-chain fatty acid (LCFA) metabolism/storage causes cardiomyopathy in animal models of type 2 diabetes. Medium-chain fatty acids (MCFAs) are absorbed and oxidized efficiently. Data in animal models of diabetes suggest MCFAs may benefit the heart.



Our objective was to test the effects of an MCFA-rich diet vs an LCFA-rich diet on plasma lipids, cardiac steatosis, and function in patients with type 2 diabetes.



This was a double-blind, randomized, 2-week matched-feeding study.



The study included ambulatory patients in the general community.



Sixteen patients, ages 37-65 years, with type 2 diabetes, an ejection fraction greater than 45%, and no other systemic disease were included.



Fourteen days of a diet rich in MCFAs or LCFAs, containing 38% as fat in total, was undertaken.



Cardiac steatosis and function were the main outcome measures, with lipidomic changes considered a secondary outcome.



The relatively load-independent measure of cardiac contractility, S', improved in the MCFA group (P < .05). Weight-adjusted stroke volume and cardiac output decreased in the LCFA group (both P < .05). The MCFA, but not the LCFA, diet decreased several plasma sphingolipids, ceramide, and acylcarnitines implicated in diabetic cardiomyopathy, and changes in several sphingolipids correlated with improved fasting insulins.



Although a diet high in MCFAs does not change cardiac steatosis, our findings suggest that the MCFA-rich diet alters the plasma lipidome and may benefit or at least not harm cardiac function and fasting insulin levels in humans with type 2 diabetes. Larger, long-term studies are needed to further evaluate these effects in less-controlled settings.

title: Increased oxysterol contents in diabetic rat hearts: their involvement in diabetic cardiomyopathy.
abstract: Abnormal lipid metabolism associated with diabetes mellitus has been proposed to be involved in the pathogenesis of diabetic cardiomyopathy. Oxysterols, oxidation derivatives of cholesterol, are known to be highly cytotoxic.



To monitor changes in myocardial oxysterols and to assess the effect of probucol, a lipid lowering agent, on myocardial lipids and oxysterols in diabetic rats.



Streptozotocin-induced diabetic rats were divided into two groups; one group was put on a standard diet, and the other a diet containing 1% (weight/weight) probucol for eight weeks. Two oxysterols, 7 beta-hydroxycholesterol and 7-ketocholesterol, were identified in myocardium by capillary gas chromatography. Both 7 beta-hydroxycholesterol and 7-ketocholesterol were significantly increased in diabetic rats (49.9 +/- 9.4 ng/mg dry weight versus 5.8 +/- 1.2 in controls, P < 0.05; and 5.3 +/- 1.2 ng/mg dry weight versus 1.7 +/- 0.1 in controls, P < 0.01, respectively). Probucol reduced not only plasma lipids but also myocardial lipids except for cholesterol and sphingomyelin fractions. However, probucol did not improve insulin deficiency, glucose metabolism or myocardial oxysterol contents.



This study demonstrated an increase in oxysterols in the myocardium of diabetic rats, suggesting that oxysterols may play a role in the development of diabetic cardiomyopathy. Probucol did not decrease the myocardial oxysterol content at the dose used in this study, suggesting that the increase in oxysterols may not be attributed to high circulating concentrations of lipids, but rather to disturbed myocardial metabolism due to insulin deficiency.

title: Substrate selection in the isolated working rat heart: effects of reperfusion, afterload, and concentration
abstract: A study of substrate selection in the isolated heart was made using13C NMR isotopomer analysis, a method that unequivocally identifies relative substrate utilization. This technique has several advantages over conventional approaches used to study this problem. It detects the labeling of metabolic end-products present in tissue, as opposed to more indirect methods such as measurement of respiratory quotient, arteriovenous differences, or specific activity changes in the added substrate. It also has advantages over methods such as14CO2 release, which may involve dilution of label with unlabeled pools before CO2 release. Furthermore, it can measure the relative oxidation of up to four substrates in a single experiment, which other labeling techniques cannot conveniently achieve. Substrates selection was considered in light of its effects on myocardial efficiency and recovery from ischemia. A mixture of four substrates (acetoacetate, glucose, lactate, and a mixture of long chain fatty acids), present at physiological concentration (0.17, 5.5, 1.2, and 0.35 mM, respectively), was examined. This is the first use of such a mixture in the study of substrate selection in an isolated organ preparation. At these concentrations, it was found that fatty acids supplied the majority of the acetyl-CoA (49%), and a substantial contribution was also provided by acetoacetate (23%). This suggests that the ketone bodies are a more important substrate than generally considered. Indeed, normalizing the relative utilizations on the basis of acetyl-CoA equivalents, ketone bodies were by far the preferred substrate. The relative lactate oxidation was only 15%, and glucose oxidation could not be detected. No change in utilization was detected after 15 min of ischemia followed by 40 min of reperfusion. The change in substrate selection with afterload was examined, to mimic the stress-related changes in workload found with ischemia. Only minor changes were found. Substrate selection from the same group of substrates, but employing concentrations observed during starvation, was also assessed. This represents the state during which most clinical treatments and evaluations are performed. In this case, acetoacetate was the most used substrate (78%), with small and equal contributions from fatty acids and endogenous substrates; the oxidation of lactate was suppressed.

title: Metabolic remodelling in diabetic cardiomyopathy
abstract: Diabetes is a risk factor for heart failure and cardiovascular mortality with specific changes to myocardial metabolism, energetics, structure, and function. The gradual impairment of insulin production and signalling in diabetes is associated with elevated plasma fatty acids and increased myocardial free fatty acid uptake and activation of the transcription factor PPARα. The increased free fatty acid uptake results in accumulation of toxic metabolites, such as ceramide and diacylglycerol, activation of protein kinase C, and elevation of uncoupling protein-3. Insulin signalling and glucose uptake/oxidation become further impaired, and mitochondrial function and ATP production become compromised. Increased oxidative stress also impairs mitochondrial function and disrupts metabolic pathways. The diabetic heart relies on free fatty acids (FFA) as the major substrate for oxidative phosphorylation and is unable to increase glucose oxidation during ischaemia or hypoxia, thereby increasing myocardial injury, especially in ageing female diabetic animals. Pharmacological activation of PPARγ in adipose tissue may lower plasma FFA and improve recovery from myocardial ischaemic injury in diabetes. Not only is the diabetic heart energetically-impaired, it also has early diastolic dysfunction and concentric remodelling. The contractile function of the diabetic myocardium negatively correlates with epicardial adipose tissue, which secretes proinflammatory cytokines, resulting in interstitial fibrosis. Novel pharmacological strategies targeting oxidative stress seem promising in preventing progression of diabetic cardiomyopathy, although clinical evidence is lacking. Metabolic agents that lower plasma FFA or glucose, including PPARγ agonism and SGLT2 inhibition, may therefore be promising options.

title: Myocardial metabolism in insulin-deficient diabetic humans without coronary artery disease.
abstract: Eleven insulin-dependent diabetes mellitus (IDDM) patients with angiographically normal coronary arteries and a normal hemodynamic response to an echocardiographic-dipyridamole test and 12 normal controls were studied at rest and after atrial pacing simultaneously sampling arterial and coronary sinus blood. In IDDM patients, despite hyperglycemia [10.0 +/- 2.0 (SE) mmol/l], myocardial glucose uptake was slightly lower than in controls. This process was significantly activated in both groups during atrial pacing. The isotopically calculated net flux of lactate across myocardium, in agreement with the net balance value based on unlabeled lactate-pyruvate arteriovenous differences, showed a net uptake in controls (3.5 +/- 0.6 mumol.min-1.1.73 m-2) and a net release in IDDM (12.4 +/- 2.6; P less than 0.01). Atrial pacing stimulated lactate uptake in both groups. Myocardial uptake of ketone bodies was significantly higher in IDDM (37.0 +/- 6.3 mumol.min-1.1.73 m-2) than in controls (10.1 +/- 3.4 mumol.min-1.1.73 m-2; P less than 0.01). Free fatty acid uptake was also significantly greater in IDDM than in controls (44.1 +/- 7.0 vs. 24.1 +/- 5.1 mumol.min-1.1.73 m-2; P less than 0.01). Alanine and branched amino acids were released by diabetic but not by control hearts at rest. The normalization of blood glucose concentrations restored normal patterns of lactate and ketone body kinetics across diabetic myocardium. In conclusion, 1) at rest, myocardial lactate and amino acid uptake is markedly impaired in IDDM without coronary artery disease, and 2) the metabolic abnormalities of the diabetic myocardium are not a primary phenomenon but rather a consequence of hypoinsulinemia and hyperglycemia because insulin administration, resulting in euglycemia, restored normal patterns of cardiac metabolism.

title: Identification of Energy Metabolism Changes in Diabetic Cardiomyopathy Rats Using a Metabonomic Approach
abstract: Diabetic cardiomyopathy (DCM) is a serious complication of diabetes. It is therefore crucial to elucidate the characteristic metabolic changes that occur during the development of diabetes to gain an understanding the pathogenesis of this disease and identify potential drug targets involved.  1H nuclear magnetic resonance-based metabonomics combined with HPLC measurements were used to determine the metabolic changes in isolated cardiac tissues after 5 weeks, 9 weeks, and 15 weeks in rats treated with streptozotocin.  Pattern recognition analysis clearly discriminated the diabetic rats from time-matched control rats, suggesting that the metabolic profile of the diabetic group was markedly different from that of the controls. Quantitative analysis showed that the levels of energy metabolites, such as the high-energy phosphate pool (ATP and creatine), significantly decreased in a time-dependent manner. Correlation analysis revealed the inhibition of glycolysis and the tricarboxylic acid (TCA) cycle, enhanced lipid metabolism, and changes in some amino acids, which may have led to the decline in energy production in the DCM rats.  The results indicated that the administration of energy substances or the manipulation of myocardial energy synthesis induced by increased glucose oxidation may contribute to the amelioration of cardiac dysfunction in diabetes.

title: Shift to Fatty Substrate Utilization in Response to Sodium–Glucose Cotransporter 2 Inhibition in Subjects Without Diabetes and Patients With Type 2 Diabetes
abstract: Pharmacologically induced glycosuria elicits adaptive responses in glucose homeostasis and hormone release. In type 2 diabetes (T2D), along with decrements in plasma glucose and insulin levels and increments in glucagon release, sodium–glucose cotransporter 2 (SGLT2) inhibitors induce stimulation of endogenous glucose production (EGP) and a suppression of tissue glucose disposal (TGD). We measured fasting and postmeal glucose fluxes in 25 subjects without diabetes using a double glucose tracer technique; in these subjects and in 66 previously reported patients with T2D, we also estimated lipolysis (from [2H5]glycerol turnover rate and circulating free fatty acids, glycerol, and triglycerides), lipid oxidation (LOx; by indirect calorimetry), and ketogenesis (from circulating β-hydroxybutyrate concentrations). In both groups, empagliflozin administration raised EGP, lowered TGD, and stimulated lipolysis, LOx, and ketogenesis. The pattern of glycosuria-induced changes was similar in subjects without diabetes and in those with T2D but quantitatively smaller in the former. With chronic (4 weeks) versus acute (first dose) drug administration, glucose flux responses were attenuated, whereas lipid responses were enhanced; in patients with T2D, fasting β-hydroxybutyrate levels rose from 246 ± 288 to 561 ± 596 µmol/L (P < 0.01). We conclude that by shunting substantial amounts of carbohydrate into urine, SGLT2-mediated glycosuria results in a progressive shift in fuel utilization toward fatty substrates. The associated hormonal milieu (lower insulin-to-glucagon ratio) favors glucose release and ketogenesis.

title: Diabetes and branched‐chain amino acids: What is the link?
abstract: Branched-chain amino acids (BCAA) have increasingly been studied as playing a role in diabetes, with the PubMed search string "diabetes" AND "branched chain amino acids" showing particular growth in studies of the topic over the past decade . In the Young Finn's Study, BCAA and, to a lesser extent, the aromatic amino acids phenylalanine and tyrosine were associated with insulin resistance (IR) in men but not in women, whereas the gluconeogenic amino acids alanine, glutamine, or glycine, and several other amino acids (i.e. histidine, arginine, and tryptophan) did not show an association with IR. Obesity may track more strongly than metabolic syndrome and diabetes with elevated BCAA. In a study of 1302 people aged 40-79; higher levels of BCAA tracked with older age, male sex, and metabolic syndrome, as well as with obesity, cardiovascular risk, dyslipidemia, hypertension, and uric acid. Medium- and long-chain acylcarnitines, by-products of mitochondrial catabolism of BCAAs, as well as branched-chain keto acids and the BCAA themselves distinguished obese people having versus not having features of IR, and in a study of 898 patients with essential hypertension, the BCAA and tyrosine and phenylalanine were associated with metabolic syndrome and impaired fasting glucose. In a meta-analysis of three genome-wide association studies, elevations in BCAA and, to a lesser extent, in alanine tracked with IR, whereas higher levels of glutamine and glycine were associated with lesser likelihood of IR. Given these associations with IR, it is not surprising that a number of studies have shown higher BCAA levels in people with and prior to development of type 2 diabetes (T2D), although this has particularly been shown in Caucasian and Asian ethnic groups while not appearing to occur in African Americans. Similarly, higher BCAA levels track with cardiovascular disease.  At this point, whether whole-body or tissue-specific BCAA metabolism is increased or decreased in states of insulin-resistant obesity and T2D is uncertain. Insulin action in the hypothalamus induces but overfeeding decreases hepatic BCKDH, leading to the concept that hypothalamic insulin resistance impairs BCAA metabolism in obesity and diabetes, so that plasma BCAAs may be markers of hypothalamic insulin action rather than direct mediators of changes in IR. A way to address this may be to understand the effects of changes in diet and other interventions on BCAA, as well as on IR and T2D. In an animal model, lowering dietary BCAA increased energy expenditure and improved insulin sensitivity. Two large human population studies showed an association of estimated dietary BCAA intake with T2D risk, although another population study showed higher dietary BCAA to be associated with lower T2D risk. Ethnic differences, reflecting underlying differences in genetic variants, may be responsible for such differences. In the study of Asghari et al. in the current issue of the Journal of Diabetes, BCAA intake was associated with the development of subsequent IR. Studies of bariatric surgery suggest lower basal and post-insulin infusion BCAA levels are associated with greater insulin sensitivity, with reductions in BCAA not seen with weight loss per se with gastric band procedures, but occurring after Roux-en-Y gastric bypass, an intervention that may have metabolic benefits over and above those from reduction in body weight. The gut microbiota may be important for the supply of the BCAA to mammalian hosts, either by de novo biosynthesis or by modifying nutrient absorption. A final fascinating preliminary set of observations is that of the effects of empagliflozin on metabolomics; evidence of increased Krebs cycle activation and of higher levels of BCAA metabolites, such as acylcarnitines, suggests that sodium-glucose cotransporter 2 (SGLT2) inhibition may, to some extent, involve BCAA metabolism. Certainly, we do not yet have a full understanding of these complex associations. However, the suggestion of multiple roles of BCAA in the development of IR promises to be important and to lead to the development of novel effective T2D therapies.

title: Human amylin proteotoxicity impairs protein biosynthesis, and alters major cellular signaling pathways in the heart, brain and liver of humanized diabetic rat model in vivo
abstract: IntroductionChronic hypersecretion of the 37 amino acid amylin is common in type 2 diabetics (T2D). Recent studies implicate human amylin aggregates cause proteotoxicity (cell death induced by misfolded proteins) in both the brain and the heart.ObjectivesIdentify systemic mechanisms/markers by which human amylin associated with cardiac and brain defects might be identified.MethodsWe investigated the metabolic consequences of amyloidogenic and cytotoxic amylin oligomers in heart, brain, liver, and plasma using non-targeted metabolomics analysis in a rat model expressing pancreatic human amylin (HIP model).ResultsFour metabolites were significantly different in three or more of the four compartments (heart, brain, liver, and plasma) in HIP rats. When compared to a T2D rat model, HIP hearts uniquely had significant DECREASES in five amino acids (lysine, alanine, tyrosine, phenylalanine, serine), with phenylalanine decreased across all four tissues investigated, including plasma. In contrast, significantly INCREASED circulating phenylalanine is reported in diabetics in multiple recent studies.ConclusionDECREASED phenylalanine may serve as a unique marker of cardiac and brain dysfunction due to hyperamylinemia that can be differentiated from alterations in T2D in the plasma. While the deficiency in phenylalanine was seen across tissues including plasma and could be monitored, reduced tyrosine was seen only in the brain. The 50 % reduction in phenylalanine and tyrosine in HIP brains is significant given their role in supporting brain chemistry as a precursor for catecholamines (dopamine, norepinephrine, epinephrine), which may contribute to the increased morbidity and mortality in diabetics at a multi-system level beyond the effects on glucose metabolism.

title: Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart
abstract: Exposure to environmental hypoxia, at high altitude or in a chamber, impairs cardiac energetics and alters mitochondrial function. Inorganic nitrate, a ubiquitous dietary constituent, improves mitochondrial efficiency, lowering the oxygen cost of exercise, whilst elevated circulating nitrogen oxide levels in high‐altitude natives enhances blood flow. Here we report that dietary nitrate supplementation prevents hypoxia‐induced changes in cardiac mitochondrial function and energetics, whilst ameliorating oxidative stress, suggesting improved tissue oxygenation. Furthermore, nitrate supplementation suppresses cardiac arginase expression and increases tissue l‐arginine levels under both hypoxic and normoxic conditions, underpinning a novel mechanism to enhance the availability of nitric oxide. Nitrate supplementation may thus be of benefit to individuals exposed to hypobaric hypoxia at altitude or in patients with diseases characterised by tissue hypoxia and energetic impairment, such as heart failure and chronic obstructive pulmonary disease, or in the critically ill.

title: Fatty Acids Prevent Hypoxia-Inducible Factor-1α Signaling Through Decreased Succinate in Diabetes
abstract: Visual

title: The mechanisms of alloxan- and streptozotocin-induced diabetes
abstract: Alloxan and streptozotocin are toxic glucose analogues that preferentially accumulate in pancreatic beta cells via the GLUT2 glucose transporter. In the presence of intracellular thiols, especially glutathione, alloxan generates reactive oxygen species (ROS) in a cyclic redox reaction with its reduction product, dialuric acid. Autoxidation of dialuric acid generates superoxide radicals, hydrogen peroxide and, in a final iron-catalysed reaction step, hydroxyl radicals. These hydroxyl radicals are ultimately responsible for the death of the beta cells, which have a particularly low antioxidative defence capacity, and the ensuing state of insulin-dependent ‘alloxan diabetes’. As a thiol reagent, alloxan also selectively inhibits glucose-induced insulin secretion through its ability to inhibit the beta cell glucose sensor glucokinase. Following its uptake into the beta cells, streptozotocin is split into its glucose and methylnitrosourea moiety. Owing to its alkylating properties, the latter modifies biological macromolecules, fragments DNA and destroys the beta cells, causing a state of insulin-dependent diabetes. The targeting of mitochondrial DNA, thereby impairing the signalling function of beta cell mitochondrial metabolism, also explains how streptozotocin is able to inhibit glucose-induced insulin secretion.

title: STZ Transport and Cytotoxicity: Specific Enhancement in GLUT2-Expressing Cells
abstract: The glucose analog streptozotocin (STZ) has long been used as a tool for creating experimental diabetes because of its relatively specific β-cell cytotoxic effect, but the mechanism by which systemic injection of STZ causes β-cell destruction is not well understood. In the current study, we have used insulinoma (RIN) and AtT-20ins cell lines engineered for overexpression of GLUT2 or GLUT1 to investigate the role of glucose transporter isoforms in mediating STZ cytotoxicity. The in vivo effects of STZ were evaluated by implantation of RIN cells expressing or lacking GLUT2 into athymic nude rats. The drug had a potent cytotoxic effect on RIN cells expressing GLUT2, but had no effect on cells lacking GLUT2 expression, as indicated by histological analysis and measurement of the blood glucose levels of treated animals. The preferential cytotoxic effect of STZ on GLUT2-expressing cell lines was confirmed by in vitro analysis of GLUT2-expressing and untransfected RIN cells, as well as GLUT2- and GLUTl-overexpressing AtT-20ins cells. Consistent with these data, only GLUT2-expressing RIN or AtT-20ins cells transported STZ efficiently. We conclude that expression of GLUT2 is required for efficient killing of neuroendocrine cells by STZ, and this effect is related to specific recognition of the drug as a transported substrate by GLUT2 but not GLUT1

title: Novel "Dual Hit" Rat Model of Diabetic Cardiomyopathy
abstract: The pathogenesis of diabetic cardiomyopathy (DC) is poorly understood and new drugs targeting the myocardium are absent. This may be ascribed to the failure of available pre-clinical models to recapitulate essential clinical features of DC and heart failure. We hypothesized that the combination of experimental diabetes and pharmacologically induced cardiac stress might provide a novel rat model displaying a distinct profile of DC. Non-insulin dependent type 1 like diabetes mellitus was induced by partial (90%) pancreatectomy (Px). Pronounced hyperglycemia was established within two weeks (blood glucose levels of 23.3 ± 3.7 mM). Five weeks post Px or sham surgery, vehicle or the sympathomimetic agent, isoproterenol (Iso, 1 mg/kg, SC) was administered for 10 days. Ten weeks after surgery, the heart was isolated for detailed histological and molecular characterization. The relative weight of the left ventricle (LV) was significantly increased in Px-Iso rats compared to sham-vehicle treated control rats (0.22 ± 0.03 vs. 0.25 ± 0.02 mg/g BW, p In conclusion, the Px-Iso rat model may provide a state-of-the-art model of DC displaying key features of the clinical condition. Hereby, this model may be useful in the evaluation of cardiovascular effects of novel compounds in the pre-clinical phase of drug development. Disclosure L. Thisted: Employee; Self; Gubra.  Stock/Shareholder; Self; Novo Nordisk A/S. Employee; Self; Gubra. Stock/Shareholder; Self; Gubra. T. Secher: Employee; Self; Gubra. Employee; Spouse/Partner; Novo Nordisk A/S. M.B. Thomsen: None. T. Jespersen: None. A.J. Murray: None.  Board Member; Self; Gubra. Employee; Self; Gubra. Stock/Shareholder; Self; Gubra. L.N. Fink: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. T.X. Pedersen: Employee; Self; Gubra.

title: Cardiac carbohydrate metabolism in Zucker diabetic fatty rats.
abstract: The aim of this study was to test the hypothesis that, shortly after the development of Type-2 diabetes, alterations in cardiac carbohydrate metabolism precede the onset of abnormalities in systolic function.



Hearts from 11-week-old Zucker diabetic fatty (ZDF) rats and age matched controls were perfused in the isovolumic Langendorff mode with 13C-labeled glucose, lactate and pyruvate and unlabeled fatty acids. 13C-Nuclear magnetic resonance glutamate isotopomer analysis was carried out to determine the contributions of substrates to energy production.



The ZDF group was hyperglycemic and the relative flux through pyruvate dehydrogenase (PDH) was significantly depressed compared to lean controls. In the lean group, lactate, pyruvate and glucose contributed 64+/-3, 24+/-3 and 11+/-1%, respectively, to total pyruvate oxidation. In the ZDF group, the contribution of glucose both to total pyruvate oxidation and to tissue lactate and alanine formation was significantly depressed. Cardiac function assessed by the rate-pressure product was similar in both groups. The fraction of active PDH was decreased in the ZDF group compared to controls (p<0.025).



These results highlight significant changes in cardiac carbohydrate metabolism shortly after the development of hyperglycemia in a model of Type 2 diabetes in the absence of overt changes in systolic function.

title: Myocardial insulin resistance associated with chronic hypertriglyceridemia and increased FFA levels in Type 2 diabetic patients.
abstract: We evaluated the influence of chronic hypertriglyceridemia and endothelial dysfunction on myocardial glucose uptake (MGU) in Type 2 diabetic patients without coronary heart disease. Patients were divided into two groups according to fasting triglyceride (TG) levels: 5.4 +/- 1.1 and 1.5 +/- 0.3 mmol/l for high- and normal-TG groups, respectively. Five subjects were assigned to the high-TG group and 11 to the normal-TG group. Age, gender, body mass index, systolic and diastolic blood pressure, glucose, insulin, HbA1c, cholesterol, and HDL cholesterol were similar in the two groups, whereas free fatty acid (FFA) levels were higher in the high-TG group basally and at the end of the clamp. Furthermore, five healthy subjects were subjected to the same protocol and used as the control group. MGU was assessed by using 18F-labeled 2-fluoro-2-deoxy-D-glucose under hyperglycemic-hyperinsulinemic conditions. Basal endothelin-1 and nitric oxide levels were significantly higher in the high-TG group than in the normal-TG and control groups, and cGMP and maximal postischemic vasodilation were significantly decreased in the high-TG group compared with the normal-TG and control groups. However, significant alterations were found in the same parameters in the normal-TG group compared with the control group. By the end of the hyperglycemic clamp, in the high-TG group, MGU was approximately 40 and 65% of that in the normal-TG and control groups. MGU negatively correlated with TG, FFA, and endothelin-1, whereas a positive correlation was found with cGMP and maximal postischemic vasodilation. In conclusion, increased TG and FFA levels are risks, in addition to Type 2 diabetes mellitus, for myocardial insulin resistance, endothelial dysfunction, and alteration of nitric oxide/cGMP levels.

title: Effect of Obesity and Insulin Resistance on Myocardial Substrate Metabolism and Efficiency in Young Women
abstract: Obesity is a risk factor for impaired cardiac performance, particularly in women. Animal studies suggest that alterations in myocardial fatty acid metabolism and efficiency in obesity can cause decreased cardiac performance. In the present study, we tested the hypothesis that myocardial fatty acid metabolism and efficiency are abnormal in obese women. We studied 31 young women (body mass index [BMI] 19 to 52 kg/m2); 19 were obese (BMI >30 kg/m2). Myocardial oxygen consumption (M&OV0312;o2) and fatty acid uptake (MFAUp), utilization (MFAU), and oxidation (MFAO) were quantified by positron emission tomography. Cardiac work was measured by echocardiography, and efficiency was calculated as work/M &OV0312;o2. BMI correlated with M &OV0312;o2 (r =0.58, P =0.0006), MFAUp (r =0.42, P <0.05), and efficiency (r = −0.40, P <0.05). Insulin resistance, quantified by the glucose area under the curve (AUC) during an oral glucose tolerance test, correlated with MFAUp (r =0.55, P <0.005), MFAU (r =0.62, P <0.001), and MFAO (r =0.58, P <0.005). A multivariate, stepwise regression analysis showed that BMI was the only independent predictor of M&OV0312;o2 and efficiency (P =0.0005 and P <0.05, respectively). Glucose AUC was the only independent predictor of MFAUp, MFAU, and MFAO (P <0.05, <0.005, and <0.005, respectively). In young women, obesity is a significant predictor of increased M&OV0312;o2 and decreased efficiency, and insulin resistance is a robust predictor of MFAUp, MFAU, and MFAO. This increase in fatty acid metabolism and decrease in efficiency is concordant with observations made in experimental models of obesity. These metabolic changes may play a role in the pathogenesis of decreased cardiac performance in obese women.

title: Trimetazidine reduces endogenous free fatty acid oxidation and improves myocardial efficiency in obese humans.
abstract: The metabolic modulator trimetazidine (TMZ) has been suggested to induce a metabolic shift from myocardial fatty acid oxidation (FAO) to glucose utilization, but this mechanism remains unproven in humans. The oxidation of plasma derived FA is commonly measured in humans, whereas the contribution of FA from triglycerides stored in the myocardium has been poorly characterized.



To verify the hypothesis that TMZ induces a metabolic shift, we combined positron emission tomography (PET) and magnetic resonance spectroscopy ((1)H-MRS) to measure myocardial FAO from plasma and intracellular lipids, and myocardial glucose metabolism. Nine obese subjects were studied before and after 1 month of TMZ treatment. Myocardial glucose and FA metabolism were assessed by PET with (18)F-fluorodeoxyglucose and (11)C-palmitate. (1)H-MRS was used to measure myocardial lipids, the latter being integrated into the PET data analysis to quantify myocardial triglyceride turnover.



Myocardial FAO derived from intracellular lipids was at least equal to that of plasma FAs (P = NS). BMI and cardiac work were positively associated with the oxidation of plasma derived FA (P ≤ 0.01). TMZ halved total and triglyceride-derived myocardial FAO (32.7 ± 8.0 to 19.6 ± 4.0 μmol/min and 23.7 ± 7.5 to 10.3 ± 2.7 μmol/min, respectively; P ≤ 0.05). These changes were accompanied by increased cardiac efficiency since unchanged LV work (1.6 ± 0.2 to 1.6 ± 0.1 Watt/g × 10(2), NS) was associated with decreased work energy from the intramyocardial triglyceride oxidation (1.6 ± 0.5 to 0.4 ± 0.1 Watt/g × 10(2), P = 0.036).



In obese subjects, we demonstrate that myocardial intracellular triglyceride oxidation significantly provides FA-derived energy for mechanical work. TMZ reduced the oxidation of triglyceride-derived myocardial FAs improving myocardial efficiency.

title: Assessment of myocardial metabolism in diabetic rats using small-animal PET: a feasibility study.
abstract: This feasibility study was undertaken to determine whether kinetic modeling in conjunction with small-animal PET could noninvasively quantify alterations in myocardial perfusion and substrate metabolism in rats.



All small-animal PET was performed on either of 2 tomographs. Myocardial blood flow and substrate metabolism were measured in 10 male Zucker diabetic fatty rats (ZDF, fa/fa) and 10 lean littermates (Lean, Fa/+) using (15)O-water, 1-(11)C-glucose, 1-(11)C-acetate, and 1-(11)C-palmitate. Animals were 12.0 +/- 1.4-wk old.



Consistent with a type 2 diabetic phenotype, the ZDF animals showed higher plasma hemoglobin A(1c), insulin, glucose, and free fatty acid (FFA) levels than their lean controls. Myocardial glucose uptake (mL/g/min) was not significantly different between the 2 groups. However, higher glucose plasma levels in the ZDF rats resulted in higher myocardial glucose utilization (nmol/g/min) (Lean, 629 +/- 785, vs. ZDF, 1,737 +/- 1,406; P = 0.06). Similarly, myocardial FFA uptake (mL/g/min) was not significantly different between the 2 groups, (Lean, 0.51 +/- 28, vs. ZDF, 0.72 +/- 0.19; P = not significant) However, due to higher FFA plasma levels, utilization and oxidation (nmol/g/min) were significantly higher in the ZDF group (Lean, 519 +/- 462, vs. ZDF, 1,623 +/- 712, P < .001; and Lean, 453 +/- 478, vs. ZDF, 1,636 +/- 730, P < .01).



Noninvasive measurements of myocardial substrate metabolism in ZDF rats using small-animal PET are consistent with the expected early metabolic abnormalities that occur in this well-characterized model of type 2 diabetes mellitus. Thus, small-animal PET demonstrates significant promise in providing a means to link the myocardial metabolic abnormalities that occur in rat of disease with the human condition.

title: Time Course of Alterations in Myocardial Glucose Utilization in the Zucker Diabetic Fatty Rat with Correlation to Gene Expression of Glucose Transporters: A Small-Animal PET Investigation
abstract: Diabetic cardiomyopathy is associated with abnormalities in glucose metabolism. We evaluated myocardial glucose metabolism in a rodent model of type 2 diabetes, namely the Zucker diabetic fatty (ZDF) rat, and validated PET measurements of glucose uptake against gene and protein expression of glucose transporters (GLUTs).  Six lean and ZDF rats underwent small-animal PET at the age of 14 wk and at the age of 19 wk. The imaging protocol consisted of a 60-min dynamic acquisition with 18F-FDG (18.5–29.6 MBq). Dynamic images were reconstructed using filtered backprojection with a 2.5 zoom on the heart and 40 frames per imaging session. PET measurements of myocardial glucose uptake (MGUp) rate and utilization were determined with an input function derived by the hybrid image–blood-sampling algorithm on recovery-corrected anterolateral myocardial regions of interest. After the PET session at week 19 (W19), hearts were extracted for gene and protein expression analysis of GLUT-1 and GLUT-4. The dependence of MGUp on gene expression of GLUT-1 and GLUT-4 was characterized by multiple-regression analysis.  MGUp in ZDF rats at both week 14 (W14) and W19 (P < 0.006) was significantly lower than MGUp in lean littermate control rats. Moreover, lean rats at W19 displayed significantly higher MGUp than they did at W14 (P = 0.007). Consistent with a diminished MGUp result, gene expression of GLUT-4 was significantly (P = 0.004) lower in ZDF rats. Finally, MGUp significantly (P = 0.0003) correlated with gene expression of GLUT-4.  Using small-animal PET, we confirmed alterations in myocardial glucose utilization and validated PET measurement of MGUp against gene and protein expression of GLUTs in the diabetic heart of an animal model of type 2 diabetes.

title: Lipid zonation and phospholipid remodeling in nonalcoholic fatty liver disease
abstract: Nonalcoholic fatty liver disease (NAFLD) can progress from simple steatosis (i.e., nonalcoholic fatty liver [NAFL]) to nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. Currently, the driver for this progression is not fully understood; in particular, it is not known how NAFLD and its early progression affects the distribution of lipids in the liver, producing lipotoxicity and inflammation. In this study, we used dietary and genetic mouse models of NAFL and NASH and translated the results to humans by correlating the spatial distribution of lipids in liver tissue with disease progression using advanced mass spectrometry imaging technology. We identified several lipids with distinct zonal distributions in control and NAFL samples and observed partial to complete loss of lipid zonation in NASH. In addition, we found increased hepatic expression of genes associated with remodeling the phospholipid membrane, release of arachidonic acid (AA) from the membrane, and production of eicosanoid species that promote inflammation and cell injury. The results of our immunohistochemistry analyses suggest that the zonal location of remodeling enzyme LPCAT2 plays a role in the change in spatial distribution for AA‐containing lipids. This results in a cycle of AA‐enrichment in pericentral hepatocytes, membrane release of AA, and generation of proinflammatory eicosanoids and may account for increased oxidative damage in pericentral regions in NASH.  NAFLD is associated not only with lipid enrichment, but also with zonal changes of specific lipids and their associated metabolic pathways. This may play a role in the heterogeneous development of NAFLD.

title: Current State and Future Challenges of Mass Spectrometry Imaging for Clinical Research.
abstract: The ability of mass spectrometry imaging (MSI) to localize panels of biomolecules in tissues, without prior knowledge of their presence and in a label-free manner, has led to a rapid and substantial impact in clinical and pharmacological research, uncovering biomolecular changes associated with disease and providing low cost imaging of pharmaceuticals. This Feature article will give an introduction to the capabilities and role of MSI in the clinical analysis of patient tissues and discusses those improvements that are necessary for the progression of MSI toward routine clinical application.

title: MALDI mass spectrometric imaging of cardiac tissue following myocardial infarction in a rat coronary artery ligation model.
abstract: Although acute myocardial infarction (MI) is consistently among the top causes of death in the United States, the spatial distribution of lipids and metabolites following MI remains to be elucidated. This work presents the investigation of an in vivo rat model of MI using mass spectrometric imaging (MSI) and multivariate data analysis. MSI was conducted on cardiac tissue following a 24-h left anterior descending coronary artery ligation to analyze multiple compound classes. First, the spatial distribution of a small metabolite, creatine, was used to identify areas of infarcted myocardium. Second, multivariate data analysis and tandem mass spectrometry were used to identify phospholipid (PL) markers of MI. A number of lysophospholipids demonstrated increased ion signal in areas of infarction. In contrast, select intact PLs demonstrated decreased ion signal in the area of infarction. The complementary nature of these two lipid classes suggests increased activity of phospholipase A(2), an enzyme that has been implicated in coronary heart disease and inflammation.