Robert E. Gerszten

The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth

Many tumour cells have elevated rates of glucose uptake but reduced rates of oxidative phosphorylation. This persistence of high lactate production by tumours in the presence of oxygen, known as aerobic glycolysis, was first noted by Otto Warburg more than 75 yr ago. How tumour cells establish this altered metabolic phenotype and whether it is essential for tumorigenesis is as yet unknown. Here we show that a single switch in a splice isoform of the glycolytic enzyme pyruvate kinase is necessary for the shift in cellular metabolism to aerobic glycolysis and that this promotes tumorigenesis. Tumour cells have been shown to express exclusively the embryonic M2 isoform of pyruvate kinase. Here we use short hairpin RNA to knockdown pyruvate kinase M2 expression in human cancer cell lines and replace it with pyruvate kinase M1. Switching pyruvate kinase expression to the M1 (adult) isoform leads to reversal of the Warburg effect, as judged by reduced lactate production and increased oxygen consumption, and this correlates with a reduced ability to form tumours in nude mouse xenografts. These results demonstrate that M2 expression is necessary for aerobic glycolysis and that this metabolic phenotype provides a selective growth advantage for tumour cells in vivo.

Metabolite profiles and the risk of developing diabetes

Emerging technologies allow the high-throughput profiling of metabolic status from a blood specimen (metabolomics). We investigated whether metabolite profiles could predict the development of diabetes. Among 2,422 normoglycemic individuals followed for 12 years, 201 developed diabetes. Amino acids, amines and other polar metabolites were profiled in baseline specimens by liquid chromatography–tandem mass spectrometry (LC-MS). Cases and controls were matched for age, body mass index and fasting glucose. Five branched-chain and aromatic amino acids had highly significant associations with future diabetes: isoleucine, leucine, valine, tyrosine and phenylalanine. A combination of three amino acids predicted future diabetes (with a more than fivefold higher risk for individuals in top quartile). The results were replicated in an independent, prospective cohort. These findings underscore the potential key role of amino acid metabolism early in the pathogenesis of diabetes and suggest that amino acid profiles could aid in diabetes risk assessment.

MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions

Monocytes contribute to the development of atherosclerotic lesions in mouse models. The chemoattractant proteins (chemokines), monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), are found in human atheroma, and mice lacking receptors for these chemokines are less susceptible to atherosclerosis and have fewer monocytes in vascular lesions. Although MCP-1 has a powerful effect on monocytes, IL-8 is thought to act predominantly on neutrophils and it is unclear howit could recruit monocytes. Here we investigate the ability of chemokines to control the interaction of monocytes under flow conditions with vascular endothelium that has been transduced to express specific leukocyte-adherence receptors. We find that MCP-1 and IL-8 can each rapidly cause rolling monocytes to adhere firmly onto monolayers expressing E-selectin, whereas related chemokines do not. These effects do not correlate with either the induction of a calcium transient or chemotaxis. We conclude that chemokines are important modulators of monocyte–endothelial interactions under flow conditions. Moreover, our finding that IL-8 is a powerful trigger for firm adhesion of monocytes to vascular endothelium reveals an unexpected role for this chemokine in monocyte recruitment.

MicroRNA-33 and the SREBP Host Genes Cooperate to Control Cholesterol Homeostasis

miR-33 in Cholesterol Control With the well-established link between serum cholesterol levels and cardiovascular disease and the availability of effective cholesterol-lowering drugs, cholesterol screening has rapidly become a routine part of health care. Yet, much remains to be learned about how cholesterol levels are regulated at the cellular level (see the Perspective by Brown et al.). Now, Najafi-Shoushtari et al. (p. 1566, published online 13 May) and Rayner et al. (p. 1570, published online 13 May) have discovered a new molecular player in cholesterol control—a small noncoding RNA that, intriguingly, is embedded within the genes coding for sterol regulatory element-binding proteins (SREBPs), transcription factors already known to regulate cholesterol levels. This microRNA, called miR-33, represses expression of the adenosine triphosphate–binding cassette transporter A1, a protein that regulates synthesis of high-density lipoprotein (HDL, or “good” cholesterol) and that helps to remove “bad” cholesterol from the blood. Reducing the levels of miR-33 in mice boosted serum HDL levels, suggesting that manipulation of this regulatory circuit might be therapeutically useful. A small noncoding RNA helps regulate cholesterol levels in mice. Proper coordination of cholesterol biosynthesis and trafficking is essential to human health. The sterol regulatory element–binding proteins (SREBPs) are key transcription regulators of genes involved in cholesterol biosynthesis and uptake. We show here that microRNAs (miR-33a/b) embedded within introns of the SREBP genes target the adenosine triphosphate–binding cassette transporter A1 (ABCA1), an important regulator of high-density lipoprotein (HDL) synthesis and reverse cholesterol transport, for posttranscriptional repression. Antisense inhibition of miR-33 in mouse and human cell lines causes up-regulation of ABCA1 expression and increased cholesterol efflux, and injection of mice on a western-type diet with locked nucleic acid–antisense oligonucleotides results in elevated plasma HDL. Our findings indicate that miR-33 acts in concert with the SREBP host genes to control cholesterol homeostasis and suggest that miR-33 may represent a therapeutic target for ameliorating cardiometabolic diseases.

Sildenafil Improves Exercise Capacity and Quality of Life in Patients With Systolic Heart Failure and Secondary Pulmonary Hypertension

Background— Patients with systolic heart failure (HF) who develop secondary pulmonary hypertension (PH) have reduced exercise capacity and increased mortality compared with HF patients without PH. We tested the hypothesis that sildenafil, an effective therapy for pulmonary arterial hypertension, would lower pulmonary vascular resistance and improve exercise capacity in patients with HF complicated by PH. Methods and Results— Thirty-four patients with symptomatic HF and PH were randomized to 12 weeks of treatment with sildenafil (25 to 75 mg orally 3 times daily) or placebo. Patients underwent cardiopulmonary exercise testing before and after treatment. The change in peak &OV0312;o2 from baseline, the primary end point, was greater in the sildenafil group (1.8±0.7 mL · kg−1 · min−1) than in the placebo group (−0.27 mL · kg−1 · min−1; P=0.02). Sildenafil reduced pulmonary vascular resistance and increased cardiac output with exercise (P<0.05 versus placebo for both) without altering pulmonary capillary wedge or mean arterial pressure, heart rate, or systemic vascular resistance. The ability of sildenafil treatment to augment peak &OV0312;o2 correlated directly with baseline resting pulmonary vascular resistance (r=0.74, P=0.002) and indirectly with baseline resting right ventricular ejection fraction (r=−0.64, P=0.01). Sildenafil treatment also was associated with improvement in 6-minute walk distance (29 m versus placebo; P=0.047) and Minnesota Living With Heart Failure score (−14 versus placebo; P=0.01). Subjects in the sildenafil group experienced fewer hospitalizations for HF and a higher incidence of headache than those in the placebo group without incurring excess serious adverse events. Conclusions— Phosphodiesterase 5 inhibition with sildenafil improves exercise capacity and quality of life in patients with systolic HF with secondary PH.

Growth Differentiation Factor 11 Is a Circulating Factor that Reverses Age-Related Cardiac Hypertrophy

The most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGF-β superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a therapeutic opportunity for cardiac aging.

Metabolomic Identification of Novel Biomarkers of Myocardial Ischemia

Background— Recognition of myocardial ischemia is critical both for the diagnosis of coronary artery disease and the selection and evaluation of therapy. Recent advances in proteomic and metabolic profiling technologies may offer the possibility of identifying novel biomarkers and pathways activated in myocardial ischemia. Methods and Results— Blood samples were obtained before and after exercise stress testing from 36 patients, 18 of whom demonstrated inducible ischemia (cases) and 18 of whom did not (controls). Plasma was fractionated by liquid chromatography, and profiling of analytes was performed with a high-sensitivity electrospray triple-quadrupole mass spectrometer under selected reaction monitoring conditions. Lactic acid and metabolites involved in skeletal muscle AMP catabolism increased after exercise in both cases and controls. In contrast, there was significant discordant regulation of multiple metabolites that either increased or decreased in cases but remained unchanged in controls. Functional pathway trend analysis with the use of novel software revealed that 6 members of the citric acid pathway were among the 23 most changed metabolites in cases (adjusted P=0.04). Furthermore, changes in 6 metabolites, including citric acid, differentiated cases from controls with a high degree of accuracy (P<0.0001; cross-validated c-statistic=0.83). Conclusions— We report the novel application of metabolomics to acute myocardial ischemia, in which we identified novel biomarkers of ischemia, and from pathway trend analysis, coordinate changes in groups of functionally related metabolites.

Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment

Leukotriene B4 (LTB4) was originally described as a potent lipid myeloid cell chemoattractant, rapidly generated from innate immune cells, that activates leukocytes through the G protein–coupled receptor BLT1. We report here that BLT1 is expressed on effector CD4+ T cells generated in vitro as well as in vivo when effector T cells migrate out of the lymphoid compartment and are recruited into peripheral tissues. BLT1 mediated LTB4-induced T helper type 1 (TH1) and TH2 cell chemotaxis and firm adhesion to endothelial cells under flow, as well as early CD4+ and CD8+ T cell recruitment into the airway in an asthma model. Our findings show that the LTB4-BLT1 pathway is involved in linking early immune system activation and early effector T cell recruitment.

Assessment of Echocardiography and Biomarkers for the Extended Prediction of Cardiotoxicity in Patients treated with Anthracyclines, Taxanes and Trastuzumab

Background—Because cancer patients survive longer, the impact of cardiotoxicity associated with the use of cancer treatments escalates. The present study investigates whether early alterations of myocardial strain and blood biomarkers predict incident cardiotoxicity in patients with breast cancer during treatment with anthracyclines, taxanes, and trastuzumab. Methods and Results—Eighty-one women with newly diagnosed human epidermal growth factor receptor 2–positive breast cancer, treated with anthracyclines followed by taxanes and trastuzumab were enrolled to be evaluated every 3 months during their cancer therapy (total of 15 months) using echocardiograms and blood samples. Left ventricular ejection fraction, peak systolic longitudinal, radial, and circumferential myocardial strain were calculated. Ultrasensitive troponin I, N-terminal pro–B-type natriuretic peptide, and the interleukin family member (ST2) were also measured. Left ventricular ejection fraction decreased (64 ± 5% to 59 ± 6%; P<0.0001) over 15 months. Twenty-six patients (32%, [22%–43%]) developed cardiotoxicity as defined by the Cardiac Review and Evaluation Committee Reviewing Trastuzumab; of these patients, 5 (6%, [2%–14%]) had symptoms of heart failure. Peak systolic longitudinal myocardial strain and ultrasensitive troponin I measured at the completion of anthracyclines treatment predicted the subsequent development of cardiotoxicity; no significant associations were observed for left ventricular ejection fraction, N-terminal pro–B-type natriuretic peptide, and ST2. Longitudinal strain was <19% in all patients who later developed heart failure. Conclusions—In patients with breast cancer treated with anthracyclines, taxanes, and trastuzumab, systolic longitudinal myocardial strain and ultrasensitive troponin I measured at the completion of anthracyclines therapy are useful in the prediction of subsequent cardiotoxicity and may help guide treatment to avoid cardiac side-effects.

Metabolic Profiling of the Human Response to a Glucose Challenge Reveals Distinct Axes of Insulin Sensitivity

Glucose ingestion after an overnight fast triggers an insulin‐dependent, homeostatic program that is altered in diabetes. The full spectrum of biochemical changes associated with this transition is currently unknown. We have developed a mass spectrometry‐based strategy to simultaneously measure 191 metabolites following glucose ingestion. In two groups of healthy individuals (n=22 and 25), 18 plasma metabolites changed reproducibly, including bile acids, urea cycle intermediates, and purine degradation products, none of which were previously linked to glucose homeostasis. The metabolite dynamics also revealed insulins known actions along four key axes—proteolysis, lipolysis, ketogenesis, and glycolysis—reflecting a switch from catabolism to anabolism. In pre‐diabetics (n=25), we observed a blunted response in all four axes that correlated with insulin resistance. Multivariate analysis revealed that declines in glycerol and leucine/isoleucine (markers of lipolysis and proteolysis, respectively) jointly provide the strongest predictor of insulin sensitivity. This observation indicates that some humans are selectively resistant to insulins suppression of proteolysis, whereas others, to insulins suppression of lipolysis. Our findings lay the groundwork for using metabolic profiling to define an individuals ‘insulin response profile’, which could have value in predicting diabetes, its complications, and in guiding therapy.