64Cu-CTS: A Promising Radiopharmaceutical for the Identification of Low-Grade Cardiac Hypoxia by PET.

UNLABELLED: The subtle hypoxia underlying chronic cardiovascular disease is an attractive target for PET imaging, but the lead hypoxia imaging agents (64)Cu-2,3-butanedione bis(N4-methylthiosemicarbazone) (ATSM) and (18)F-fluoromisonidazole are trapped only at extreme levels of hypoxia and hence are insufficiently sensitive for this purpose. We have therefore sought an analog of (64)Cu-ATSM better suited to identify compromised but salvageable myocardium, and we validated it using parallel biomarkers of cardiac energetics comparable to those observed in chronic cardiac ischemic syndromes. METHODS: Rat hearts were perfused with aerobic buffer for 20 min, followed by a range of hypoxic buffers (using a computer-controlled gas mixer) for 45 min. Contractility was monitored by intraventricular balloon, energetics by (31)P nuclear MR spectroscopy, lactate and creatine kinase release spectrophotometrically, and hypoxia-inducible factor 1-α by Western blotting. RESULTS: We identified a key hypoxia threshold at a 30% buffer O2 saturation that induces a stable and potentially survivable functional and energetic compromise: left ventricular developed pressure was depressed by 20%, and cardiac phosphocreatine was depleted by 65.5% ± 14% (P < 0.05 vs. control), but adenosine triphosphate levels were maintained. Lactate release was elevated (0.21 ± 0.067 mmol/L/min vs. 0.056 ± 0.01 mmol/L/min, P < 0.05) but not maximal (0.46 ± 0.117 mmol/L/min), indicating residual oxidative metabolic capacity. Hypoxia-inducible factor 1-α was elevated but not maximal. At this key threshold, (64)Cu-2,3-pentanedione bis(thiosemicarbazone) (CTS) selectively deposited significantly more (64)Cu than any other tracer we examined (61.8% ± 9.6% injected dose vs. 29.4% ± 9.5% for (64)Cu-ATSM, P < 0.05). CONCLUSION: The hypoxic threshold that induced survivable metabolic and functional compromise was 30% O2. At this threshold, only (64)Cu-CTS delivered a hypoxic-to-normoxic contrast of 3:1, and it therefore warrants in vivo evaluation for imaging chronic cardiac ischemic syndromes.

2-methoxyestradiol and disorders of female reproductive tissues.

2-Methoxyestradiol (2ME) is an endogenous metabolite of 17ß-estradiol. Once thought of as a mere degradation product, 2ME has gained attention as an important component of reproductive physiology and as a therapeutic agent in reproductive pathologies such as preeclampsia, endometriosis, infertility, and cancer. In this review, we discuss the involvement of 2ME in reproductive pathophysiology and summarize its known mechanisms of action: microtubule disruption, inhibition of angiogenesis and stimulation of apoptosis. Currently, the clinical uses of 2ME as a single agent are limited due to its poor water solubility and thus low bioavailability; however, 2ME analogs and derivatives have been recently developed and tested as cancer treatments. Despite some isolated success stories and ongoing research, 2ME derivatives have not yet provided the expected results. The adjuvant use of 2ME derivatives with chemotherapeutic agents is hindered by their intrinsic toxicity confounding the unwanted secondary effects of chemotherapy. However, due to the well-tested tolerance of the body to high doses of native 2ME, it may the combination of native 2ME with conventional treatments that will offer novel clinically relevant regimens for cancer and other reproductive disorders.

Cardiac hypoxia imaging: second-generation analogues of 64Cu-ATSM.

UNLABELLED: Myocardial hypoxia is an attractive target for diagnostic and prognostic imaging, but current approaches are insufficiently sensitive for clinical use. The PET tracer copper(II)-diacetyl-bis(N4-methylthiosemicarbazone) ((64)Cu-ATSM) has promise, but its selectivity and sensitivity could be improved by structural modification. We have therefore evaluated a range of (64)Cu-ATSM analogs for imaging hypoxic myocardium. METHODS: Isolated rat hearts (n = 5/group) were perfused with normoxic buffer for 30 min and then hypoxic buffer for 45 min within a custom-built triple-γ-detector system to quantify radiotracer infusion, hypoxia-dependent cardiac uptake, and washout. A 1-MBq bolus of each candidate tracer (and (18)F-fluoromisonidazole for comparative purposes) was injected into the arterial line during normoxia, and during early and late hypoxia, and their hypoxia selectivity and pharmacokinetics were evaluated. The in vivo pharmacokinetics of promising candidates in healthy rats were then assessed by PET imaging and biodistribution. RESULTS: All tested analogs exhibited hypoxia sensitivity within 5 min. Complexes less lipophilic than (64)Cu-ATSM provided significant gains in hypoxic-to-normoxic contrast (14:1 for (64)Cu-2,3-butanedione bis(thiosemicarbazone) (ATS), 17:1 for (64)Cu-2,3-pentanedione bis(thiosemicarbazone) (CTS), 8:1 for (64)Cu-ATSM, P < 0.05). Hypoxic first-pass uptake was 78.2% ± 7.2% for (64)Cu-ATS and 70.7% ± 14.5% for (64)Cu-CTS, compared with 63.9% ± 11.7% for (64)Cu-ATSM. Cardiac retention of (18)F-fluoromisonidazole increased from 0.44% ± 0.17% during normoxia to 2.24% ± 0.08% during hypoxia. In vivo, normoxic cardiac retention of (64)Cu-CTS was significantly lower than that of (64)Cu-ATSM and (64)Cu-ATS (0.13% ± 0.02% vs. 0.25% ± 0.04% and 0.24% ± 0.03% injected dose, P < 0.05), with retention of all 3 tracers falling to less than 0.7% injected dose within 6 min. (64)Cu-CTS also exhibited lower uptake in liver and lung. CONCLUSION: (64)Cu-ATS and (64)Cu-CTS exhibit better cardiac hypoxia selectivity and imaging characteristics than the current lead hypoxia tracers, (64)Cu-ATSM and (18)F-fluoromisonidazole.

Assessing radiotracer kinetics in the Langendorff perfused heart.

BACKGROUND: The Langendorff perfused heart is a physiologically relevant and controllable model with potential for assessing the pharmacokinetics of new radiotracers under a range of pathophysiological conditions.. We assess the feasibility of extending the methods validated for in vivo PET data analysis to the characterisation of PET tracer kinetics applied to Langendorff perfused hearts. METHODS: Monte Carlo simulations were used to study the accuracy and reproducibility of linear and non-linear spectral analysis (SA/NLSA), the Patlak graphical method and normalised tissue activity (NA). The methods were used to analyse time-activity curves of two widely used PET tracers, [18 F]-FDG and [18 F]-FMISO, acquired ex vivo from Langendorff perfused rat hearts under normoxic and hypoxic conditions. RESULTS: Monte Carlo simulations showed NLSA to be superior to SA in identifying and quantifying the presence of irreversible trapping component (αo), for low values of αo. The performance of NLSA and SA for high values of trapping was comparable. NLSA was also more precise than SA in determining the absence of trapping over the range of simulated kinetics and SNR. Simulations also suggest that the semi-quantitative method NA is adequate for the evaluation of trapping, and it was found to be more accurate than Patlak. The values of α0 estimated with NLSA from the time series of both [18 F]-FDG and [18 F]-FMISO increased significantly from normoxia to hypoxia in agreement with previous studies. The values of trapping derived using SA increased but not significantly, reflecting the larger error associate with this method. Patlak estimated from the experimental datasets increased from normoxia to hypoxia but was not significant. NA estimated from the [18 F]-FDG data increased from normoxia to hypoxia, but was not significant, whilst NA calculated for [18 F]-FMISO time-activity curves increased significantly. CONCLUSIONS: Monte Carlo simulations suggested that spectral-based quantitative analysis methods are adequate for the kinetic characterisation of time-activity curves acquired ex vivo from perfused hearts. The uptake rate Patlak and the index NA also represent a good alternative to the SA and NLSA algorithms when the aim of the kinetic analysis is to measure changes in the amount of tracer trapped in the irreversible compartment in response to external stimuli. For low levels of trapping, NLSA and NA were subject to lower errors than SA and Patlak, respectively.

Trapped platelets activated in ischemia initiate ventricular fibrillation.

BACKGROUND: We tested the hypothesis that ischemia-induced ventricular fibrillation (VF) is facilitated by platelets, trapped regionally in the ischemic zone and activated to release arrhythmogenic secretome. METHODS AND RESULTS: In a randomized study in blood-free, buffer-perfused isolated rat hearts, ischemic zone territory (34±1% of left ventricle) was selected so that ischemia evoked VF in only 42% of controls. VF incidence was increased to 91% (P<0.05) by coronary ligation-induced trapping of freshly prepared autologous platelets (infused before and during coronary ligation, with trapping confirmed by 111In-labeled platelet autoradiographic imaging). Trapping of platelet secretome prepared ex vivo, or platelet-sized fluorospheres, did not increase ischemia-induced VF incidence. Secretome alone did, however, evoke VF in 2 sham coronary-ligated hearts. Perfusion did not activate infused platelets in sham coronary-ligated hearts, whereas ligation activated trapped platelets (assessed by thromboxane release). In a separate study, trapping whole-heparinized blood mimicked the ability of trapped platelets to increase VF incidence. This effect was not prevented by >5 days oral pretreatment in vivo with clopidogrel (10 mg/kg per day) or indomethacin (2.4 mg/kg per day). CONCLUSIONS: Platelets facilitate VF during acute ischemia independently of their ability to participate in occlusive thrombosis. Moreover, the effect is unresponsive to antiplatelet drugs commonly used. Labile secretome constituents appear to be responsible. This opens a novel avenue for antiarrhythmic drug research.

Demonstration of the retention of 64Cu-ATSM in cardiac myocytes using a novel incubation chamber for screening hypoxia-dependent radiotracers.

OBJECTIVE: We have designed a low-cost, reusable incubation system that allows cells to be cultured in either plated or suspension culture under complete atmospheric control for radiotracer characterization. We demonstrate its utility here in the first quantification of the hypoxia-dependent accumulation of Cu-diacetyl bis(N4-methylthiosemicarbazone) (Cu-ATSM) in adult rat ventricular myocytes (ARVMs). MATERIALS AND METHODS: ARVMs were allowed to adhere overnight in 9 cm culture plates (2×10 cells/dish) or were used in suspension culture, placed inside the chamber and equilibrated with either oxic (95 or 21% O2/5% CO2) or anoxic gas (95% N2/5% CO2). Cu-ATSM of 100 kBq was administered, and the cells were incubated for 30 or 60 min. Cells were then harvested, counted and fractionated to determine intracellular Cu biodistribution. RESULTS: After 1 h, the average cellular Cu retention in plated ARVMs under oxygenated conditions was 23.9 ± 2.5 mBq/cell (95% O2), increasing to 27.3 ± 5.1 under 21% O2 (P<0.05) and to 36.1 ± 3.1 under 0% O2 (P<0.05). When ARVMs were cultured in suspension, normoxic-hypoxic contrast was less marked but still significant [63.2 ± 14.1 vs. 53.4 ± 10.9% mBq/cell after 30 min (P<0.05)]. Sixty percent of tracer accumulated in the cytosol, and, although total cellular retention increased during hypoxia, there was no enrichment in any particular cellular compartment. CONCLUSION: This apparatus allows the conduction of radiotracer uptake studies in cells under complete atmospheric control, as evidenced by our first demonstration of the hypoxia-dependent uptake of Cu-ATSM in ventricular myocytes. It is ideally suited for screening, validating and characterizing novel hypoxia-selective radiotracers.

Insulin regulates GLUT1-mediated glucose transport in MG-63 human osteosarcoma cells.

Osteosarcoma is the most common type of malignant bone cancer, accounting for 35% of primary bone malignancies. Because cancer cells utilize glucose as their primary energy substrate, the expression and regulation of glucose transporters (GLUT) may be important in tumor development and progression. GLUT expression has not been studied previously in human osteosarcoma cell lines. Furthermore, although insulin and insulin-like growth factor (IGF-I) play an important role in cell proliferation and tumor progression, the role of these hormones on GLUT expression and glucose uptake, and their possible relation to osteosarcoma, have also not been studied. We determined the effect of insulin and IGF-I on GLUT expression and glucose transport in three well-characterized human osteosarcoma cell lines (MG-63, SaOs-2, and U2-Os) using immunocytochemical, RT-PCR and functional kinetic analyses. Furthermore we also studied GLUT isoform expression in osteosarcoma primary tumors and metastases by in situ hybridization and immunohistochemical analyses. RT-PCR and immunostaining show that GLUT1 is the main isoform expressed in the cell lines and tissues studied, respectively. Immunocytochemical analysis shows that although insulin does not affect levels of GLUT1 expression it does induce a translocation of the transporter to the plasma membrane. This translocation is associated with increased transport of glucose into the cell. GLUT1 is the main glucose transporter expressed in osteosarcoma, furthermore, this transporter is regulated by insulin in human MG-63 cells. One possible mechanism through which insulin is involved in cancer progression is by increasing the amount of glucose available to the cancer cell.

Glucose transporters in sex steroid hormone related cancer.

Cancer cells, as with most mammalian cells, depend on a continuous supply of glucose; not only as a precursor of glycoproteins, triglycerides and glycogen, but also as an important source of energy. This review concentrates on GLUT transporter expression in both normal and cancerous classical sex-steroid hormone tissues (i.e. breast, uterus, ovary, testis and prostate, among others). Given the importance of estrogen, progesterone and androgens in carcinogenesis, as well as in survival and propagation of these cancers, this review also highlights the current literature on hormone regulation of glucose transporters and on the role of hypoxia in their expression. Given the recent explosion of information on the newer GLUT6-12 family members, a brief overview on their function and general expression has been included. Finally, an insight into the use of glucose transporters as markers of cancer progression and clinical outcome is also discussed.

Apical and basolateral localisation of GLUT2 transporters in human lung epithelial cells.

Glucose concentrations of normal human airway surface liquid are approximately 12.5 times lower than blood glucose concentrations indicating that glucose uptake by epithelial cells may play a role in maintaining lung glucose homeostasis. We have therefore investigated potential glucose uptake mechanisms in non-polarised and polarised H441 human airway epithelial cells and bronchial biopsies. We detected mRNA and protein for glucose transporter type 2 (GLUT2) and glucose transporter type 4 (GLUT4) in non-polarised cells but GLUT4 was not detected in the plasma membrane. In polarised cells, GLUT2 protein was detected in both apical and basolateral membranes. Furthermore, GLUT2 protein was localised to epithelial cells of human bronchial mucosa biopsies. In non-polarised H441 cells, uptake of D: -glucose and deoxyglucose was similar. Uptake of both was inhibited by phloretin indicating that glucose uptake was via GLUT-mediated transport. Phloretin-sensitive transport remained the predominant route for glucose uptake across apical and basolateral membranes of polarised cells and was maximal at 5-10 mM glucose. We could not conclusively demonstrate sodium/glucose transporter-mediated transport in non-polarised or polarised cells. Our study provides the first evidence that glucose transport in human airway epithelial cells in vitro and in vivo utilises GLUT2 transporters. We speculate that these transporters could contribute to glucose uptake/homeostasis in the human airway.

Regulation of GLUT3 and glucose uptake by the cAMP signalling pathway in the breast cancer cell line ZR-75.

Increased glucose uptake as a principal energy source is a requirement for the continued survival of tumour cells. Facilitative glucose transporter-1 (GLUT1) and -3 (GLUT3) have been previously shown to be present and regulated in breast cancer cells and are associated with poor patient prognosis. In cancer cells, the cAMP secondary messenger pathway is known to potentiate described glucose transporter activators and regulate cell fate. However, no regulation of the glucose transporters in breast cancer cells by cAMP has previously been examined. Herein, we determined in the well-characterized breast cancer cell line ZR-75, if the cAMP analogue 8-br-cAMP was capable of regulating GLUT1 and GLUT3 expression and thus glucose uptake. We demonstrated that 8-br-cAMP transiently up-regulates GLUT3 mRNA levels. The use of actinomycin-D and the cloning of 1,200 bp upstream of the human GLUT3 promoter demonstrated that this regulation was transcriptional. Immunocytochemistry and Western blotting confirmed that the increase in mRNA was reflected by an increase in protein levels. No notable regulation of GLUT1 in the presence of 8-br-cAMP was detected. Finally, we determined using the non-metabolizable glucose analogue 2-DOG if this up-regulation in GLUT3 increased glucose uptake. We observed the presence of two uptake components, one corresponding to the Km of GLUT1/4 and the other to GLUT3. A doubling in the uptake velocity was observed only at the Km corresponding to GLUT3. In conclusion, we demonstrate and characterize for the first time, an up-regulation of GLUT3 mRNA, protein and glucose uptake by the cAMP pathway in breast cancer cells.

Mitochondrial uncoupling, with low concentration FCCP, induces ROS-dependent cardioprotection independent of KATP channel activation.

OBJECTIVE: Both K(ATP) channel opening drugs and ischaemic preconditioning have been suggested to protect the ischaemic heart by acting on K(ATP) channels in the inner mitochondrial membrane, uncoupling the proton gradient and partially dissipating the mitochondrial membrane potential. The aim of these studies was to use low concentrations of FCCP, a mitochondrial protonophore, to bypass the mitochondrial K(ATP) channel and partially uncouple the mitochondria and establish whether this activates protective pathways within the rat heart analogous to K(ATP) channel openers or preconditioning. METHODS: Isolated, Langendorff-perfused rat hearts were subjected to 25 min global zero-flow ischaemia and functional recovery assessed. Hearts were pretreated with FCCP (30-300 nM) in the presence or absence of glibenclamide (1 microM), 5-hydroxydecanoate (5-HD: 100 microM), N-acetyl cysteine (4 mM), or N-2-mercaptopropionyl glycine (1 mM). The metabolic consequences of FCCP perfusion in isolated hearts were studied using (31)P NMR, and reactive oxygen species (ROS) production was measured using DCF fluorescence in isolated rat ventricular myocytes. RESULTS: FCCP exerted a dose-dependent cardioprotective effect, with 100 nM FCCP being the optimal concentration. This effect could not be blocked by glibenclamide or 5-HD, but was completely attenuated by N-acetyl cysteine and N-2-mercaptopropionyl glycine. Perfusion with FCCP (100 nM) did not deplete bulk ATP during the pretreatment period but significantly depleted phosphocreatine. In ventricular myocytes, FCCP caused an antioxidant-sensitive increase in ROS production but diazoxide was without effect. CONCLUSIONS: In the isolated rat heart, partial mitochondrial uncoupling with low-dose FCCP significantly improves post-ischaemic functional recovery via a ROS-dependent pathway. This cardioprotection is not mediated via the depletion of cellular ATP or mitochondrial K(ATP) channel activation.

Differential subcellular distribution of glucose transporters GLUT1-6 and GLUT9 in human cancer: ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues.

It has been proposed that the enhanced metabolic activity of tumor cells is accompanied by an increased expression of facilitative hexose transporters (GLUTs). However, a previous immunohistochemical analysis of GLUT1 expression in 154 malignant human neoplasms failed to detect the GLUT1 isoform in 87 tumors. We used 146 normal human tissues and 215 tumor samples to reassess GLUT1 expression. A similar number of samples were used to compare the expression of GLUT2-6 and 9. The classical expression of GLUT1-5 in different normal human tissues was confirmed, however, we were unable to detect GLUT2 in human pancreatic islet cells. GLUT6 was principally detected in testis germinal cells and GLUT9 was localized in kidney, liver, heart, and adrenal. In tumor samples, GLUT1, 2, and 5 were the main transporters detected. GLUT1 was the most widely expressed transporter, however, 42% of the samples had very low-to-negative expression levels. GLUT2 was detected in 31% of the samples, being mainly expressed in breast, colon, and liver carcinoma. GLUT5 was detected in 27% of breast and colon adenocarcinoma, liver carcinoma, lymphomas, and testis seminoma samples. In situ RT-PCR and ultrastructural immunohistochemistry confirmed GLUT5 expression in breast cancer. GLUT6 and 9 are not clearly over-expressed in human cancer. The extensive expression of GLUT2 and 5 (glucose/fructose and fructose transporters, respectively) in malignant human tissues indicates that fructose may be a good energy substrate in tumor cells. Our functional data obtained in vitro in different tumor cells support this hypothesis. Additionally, these results suggest that fructose uptake could be used for positron emission tomography imaging and, may possibly represent a novel target for the development of therapeutic agents in different human cancers.

Differential regulation of glucose transporter expression by estrogen and progesterone in Ishikawa endometrial cancer cells.

Estrogen replacement therapy and other unopposed estrogen treatments increase the incidence of endometrial abnormalities, including cancer. However, this effect is counteracted by the co-administration of progesterone. In the endometrium, glucose transporter (GLUT) expression and glucose transport are known to fluctuate throughout the menstrual cycle. Here, we determined the effect of estrogen and progesterone on the expression of GLUT1-4 and on the transport of deoxyglucose in Ishikawa endometrial cancer cells. Cells were incubated with estrogen, progesterone or combined estrogen and progesterone for 24 h and the effect on the expression of GLUT1-4 and on deoxyglucose transport was determined. We show that GLUT1 expression is upregulated by estrogen and progesterone individually, but that combined estrogen and progesterone treatment reverses this increase. Hormonal treatments do not affect GLUT2, GLUT3 or GLUT4 expression. Transport studies demonstrate that estrogen increases deoxyglucose transport at Michaelis-Menten constants (Kms) corresponding to GLUT1/4, an effect which disappears when progesterone is added concomitantly. These data demonstrate that different hormonal treatments differentially regulate GLUT expression and glucose transport in this endometrial cancer cell line. This regulation mirrors the role played by estrogen and progesterone on the incidence of cancer in this tissue and suggests that GLUT1 may be utilized by endometrial cancer cells to fuel their demand for increased energy requirement.

Tissue-specific differences in 2-fluoro-2-deoxyglucose metabolism beyond FDG-6-P: a 19F NMR spectroscopy study in the rat.

2-Fluoro-[(18)F]-2-deoxy-glucose (FDG) is a positron-emitting analogue of glucose used clinically in positron emission tomography (PET) to assess glucose utilization in diseased and healthy tissue. Originally developed to measure local cerebral glucose utilization rates, it has now found applications in tumour diagnosis and in the study of myocardial glucose uptake. Once taken up into the cell, FDG is phosphorylated to FDG-6-phosphate (FDG-6-P) by hexokinase and was originally believed to be trapped as a terminal metabolite. This 'metabolic trapping' of FDG-6-P forms the basis of the analysis of PET data. In this study, we have used (19)F NMR spectroscopy to investigate FDG metabolism following the injection of a bolus of the glucose tracer into the rat (n=6). Ninety minutes after the (19)FDG injection, the brain, heart, liver and kidneys were removed and the (19)FDG metabolites in each were extracted and quantified. We report that significant metabolism of FDG occurs beyond FDG-6-P in all organs examined and that the extent of this metabolism varies from tissue to tissue (degree of metabolism beyond FDG-6-P, expressed as percentage of total organ FDG content, was brain 45 +/- 3%; heart 29 +/- 2%; liver 22+/-3% and kidney 17 +/- 3%, mean +/- SEM n=6). Furthermore, we demonstrate that the relative accumulation of each metabolite was tissue-dependent and reflected the metabolic and regulatory characteristics of each organ. Such inter-tissue differences may have implications for the mathematical modelling of glucose uptake and phosphorylation using FDG as a glucose tracer.

The action of ovarian hormones in cardiovascular disease.

The incidence of cardiovascular disease (CAD) differs between men and women, in part because of differences in risk factors and hormones. This sexual dimorphism means a lower incidence in atherosclerotic diseases in premenopausal women, which subsequently rises in postmenopausal women to eventually equal that of men. These observations point towards estrogen and progesterone playing a lifetime protective role against CAD in women. As exogenous estrogen and estrogen plus progesterone preparations produce significant reductions in low-density lipoprotein (LDL) cholesterol levels and significant increases in high-density lipoprotein (HDL) cholesterol, this should in theory lower the risk of CAD. However, results from oral contraceptive (OC) use and combined estrogen and progesterone hormone replacement therapy (HRT) have suggested that hormone replacement regimes do not provide cardiovascular protection. In fact, depending on the preparation and the presence or absence of genetic risk factors, an increased risk of cardiovascular diseases such as venous thrombosis, myocardial infarction (MI) and stroke have been observed. Interestingly, in the majority of studies the increase in risk was highest in the first year, after which an increase in risk was not observed, and in some studies a lower risk of CAD was evident after four or five years of exogenous hormone administration. While the debate continues about the merits of HRT, and several good reviews exist on the statistics of CAD in relation to exogenous hormones, we have decided to review the literature to piece together the physiological actions of estrogen and progesterone preparations on the individual mechanistic components leading to CAD; namely, the altered endothelium and the haemostatic balance between coagulation and fibrinolysis. We present possible mechanisms for how HRT and OCs protect against MI in the absence of cardiovascular risk factors but increase the incidence of MI in their presence. We also speculate on the roles played by hormones on the short- and long-term risks of cardiovascular disease.

Estrogen and progesterone up-regulate glucose transporter expression in ZR-75-1 human breast cancer cells.

Breast cancer incidence increases in women receiving combined estrogen and progesterone therapy. Breast tumors show increased expression of the glucose transporter GLUT1. We determined the effect of these hormones on GLUT1-4 expression and deoxyglucose transport in ZR-75-1 breast cancer cells. Immunoblotting, immunocytochemistry, flow cytometry, and RT-PCR showed that GLUT1 expression is up-regulated by progesterone and, to a greater degree, combined therapy. GLUT2 expression is unaffected by hormonal treatment. GLUT3 protein and RNA is up-regulated by progesterone and combined therapy, and GLUT4 protein expression is up-regulated by all hormonal treatments. Deoxyglucose transport studies revealed the presence of three transport components with characteristics corresponding to GLUT1/4, GLUT2, and GLUT3. 17beta-Estradiol produced a slight increase in transport at the Michaelis constant (Km) corresponding to GLUT3. Progesterone produced a small increase in transport at the Km corresponding to GLUT1/4, and combined 17beta-estradiol and progesterone produced a small increase in transport at the Km corresponding to GLUT3 and a large increase in transport at the Km corresponding to GLUT1/4. This indicates that 17beta-estradiol and progesterone differentially regulate GLUT1-4 expression and that these changes correlate to changes in glucose uptake. We postulate that combined hormone replacement therapy provides a survival advantage to developing ZR-75 breast cancer cells.

The action of ovarian hormones in cardiovascular disease

The incidence of cardiovascular disease (CAD) differs between men and women, in part because of differences in risk factors and hormones. This sexual dimorphism means a lower incidence in atherosclerotic diseases in premenopausal women, which subsequently rises in postmenopausal women to eventually equal that of men. These observations point towards estrogen and progesterone playing a lifetime protective role against CAD in women. As exogenous estrogen and estrogen plus progesterone preparations produce significant reductions in low-density lipoprotein (LDL) cholesterol levels and significant increases in high-density lipoprotein (HDL) cholesterol, this should in theory lower the risk of CAD. However, results from oral contraceptive (OC) use and combined estrogen and progesterone hormone replacement therapy (HRT) have suggested that hormone replacement regimes do not provide cardiovascular protection. In fact, depending on the preparation and the presence or absence of genetic risk factors, an increased risk of cardiovascular diseases such as venous thrombosis, myocardial infarction (MI) and stroke have been observed. Interestingly, in the majority of studies the increase in risk was highest in the first year, after which an increase in risk was not observed, and in some studies a lower risk of CAD was evident after four or five years of exogenous hormone administration. While the debate continues about the merits of HRT, and several good reviews exist on the statistics of CAD in relation to exogenous hormones, we have decided to review the literature to piece together the physiological actions of estrogen and progesterone preparations on the individual mechanistic components leading to CAD; namely, the altered endothelium and the haemostatic balance between coagulation and fibrinolysis. We present possible mechanisms for how HRT and OCs protect against MI in the absence of cardiovascular risk factors but increase the incidence of MI in their presence. We also speculate on the roles played by hormones on the short- and long-term risks of cardiovascular disease.

Dissociation of glucose tracer uptake and glucose transporter distribution in the regionally ischaemic isolated rat heart: application of a new autoradiographic technique.

Fluorine-18 fluoro-2-deoxyglucose ((18)FDG) and carbon-14 2-deoxyglucose ((14)C-2-DG) are both widely used tracers of myocardial glucose uptake and phosphorylation. We have recently shown, using positron emission tomography (PET) and nuclear magnetic resonance, that ischaemia-reperfusion (I-R) causes differential changes in their uptake. We describe here the novel application of an autoradiographic technique allowing the investigation of this phenomenon at high resolution, using tracer concentrations of both analogues in the dual-perfused isolated rat heart. We also investigate the importance of glucose transporter (GLUT 1 and GLUT 4) distribution in governing the observed phosphorylated analogue accumulation. Hearts ( n=5) were perfused with Krebs buffer for 40 min, made regionally zero-flow ischaemic for 40 min and reperfused for 60 min with Krebs containing tracer (18)FDG (200 MBq) and tracer (14)C-2-DG (0.37 MBq). Hearts were then frozen and five sections (10 micro m) were cut per heart, fixed and exposed on phosphor storage plates for 18 h (for (18)FDG) and then for a further 9 days (for (14)C-2-DG). Quantitative digital images of tracer accumulation were obtained using a phosphor plate reader. The protocol was repeated in a second group of hearts and GLUT 1 and GLUT 4 distribution analysed. Post-ischaemic accumulation of (18)FDG-6-P was inhibited by 38.2%+/-1.7% and (14)C-DG-6-P by 19.0%+/-2.2%, compared with control ( P<0.05). After placing seven "lines of interrogation" across each heart section and analysing the phosphorylated tracer accumulation along them, a transmural gradient of both tracers was observed; this was highest at the endocardium and lowest at the epicardium. GLUT 4 translocated to the sarcolemma in the ischaemic/reperfused region (from 24%+/-3% to 59%+/-5%), while there was no cellular redistribution of GLUT 1. We conclude that since decreased phosphorylated tracer accumulation occurs after ischaemia-reperfusion, despite greater externalisation of GLUT 4, hexokinase or the affinities of the GLUT transporters are changed under these conditions.

Glucose transporters: expression, regulation and cancer.

Mammalian cells depend on glucose as a major substrate for energy production. Glucose is transported into the cell via facilitative glucose transporters (GLUT) present in all cell types. Many GLUT isoforms have been described and their expression is cell-specific and subject to hormonal and environmental control. The kinetic properties and substrate specificities of the different isoforms are specifically suited to the energy requirements of the particular cell types. Due to the ubiquitousness of these transporters, their differential expression is involved in various disease states such as diabetes, ischemia and cancer. The majority of cancers and isolated cancer cell lines over-express the GLUT family members which are present in the respective tissue of origin under non-cancerous conditions. Moreover, due to the requirement of energy to feed uncontrolled proliferation, cancer cells often express GLUTs which under normal conditions would not be present in these tissues. This over-expression is predominantly associated with the likelihood of metastasis and hence poor patient prognosis. This article presents a review of the current literature on the regulation and expression of GLUT family members and has compiled clinical and research data on GLUT expression in human cancers and in isolated human cancer cell lines.

Lactate-induced translocation of GLUT1 and GLUT4 is not mediated by the phosphatidyl-inositol-3-kinase pathway in the rat heart.

We have determined the effect of lactate on the translocation of GLUT1 and GLUT4 and on the myocardial uptake and phosphorylation of the glucose analogues 2-deoxy-D-glucose (DG) and 2-18F-fluoro-2-deoxy-D-glucose (18FDG). The involvement of phosphatidyl-inositol-3-kinase (PI3K) in this translocation was determined using wortmannin. Hearts from fed and fasted male Wistar rats were perfused in the presence of 11 mM glucose +/- 10 mM lactate for two hours and the distribution of glucose transporters was determined using Western blot techniques. Two other groups of hearts from fed animals were perfused in the presence of 11 mM glucose +/- 10 mM lactate for two hours followed by perfusion for a further 30 minutes in the presence of 4 mM 2-deoxy-D-glucose. Using 31P NMR spectroscopy, the accumulation of 2-deoxy-D-glucose-6-phosphate (DG6P) was monitored over time. Another group of hearts from fed animals was initially perfused in the presence of 11 mM glucose for 100 minutes and then the perfusate was changed to 11 mM glucose + 10 mM lactate for a further 120 minutes. Using PET, the accumulation of 2-18F-fluoro-deoxy-D-glucose-6-phosphate (18FDG6P) was monitored throughout the whole protocol. Lactate induced the translocation of both GLUT1 and GLUT4 to the plasma membrane (from 67 +/- 1% to 82 +/- 2% and from 16 +/- 1% to 28 +/- 2%, respectively (P < 0.05)) in hearts from fed animals; similar translocations were observed in hearts from fasted animals. Wortmannin did not inhibit the translocation of either GLUT1 or GLUT4. Glucose transporter translocation was accompanied by a significant inhibition of DG6P accumulation (4.24 +/- 0.68 vs. 1.50 +/- 0.38; P < 0.001) and a decrease in the rate of 18FDG6P accumulation. In conclusion, lactate causes translocation of GLUT1 and GLUT4 to the plasma membrane, via a non-PI3K-mediated pathway. Despite this externalisation of the GLUT transporters, a marked decrease in the accumulation of both DG6P and 18FDG6P was observed.