Radiopharmacological evaluation of 6-deoxy-6-[18F]fluoro-D-fructose as a radiotracer for PET imaging of GLUT5 in breast cancer.

INTRODUCTION: Several clinical studies have shown low or no expression of GLUT1 in breast cancer patients, which may account for the low clinical specificity and sensitivity of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) used in positron emission tomography (PET). Therefore, it has been proposed that other tumor characteristics such as the high expression of GLUT2 and GLUT5 in many breast tumors could be used to develop alternative strategies to detect breast cancer. Here we have studied the in vitro and in vivo radiopharmacological profile of 6-deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF) as a potential PET radiotracer to image GLUT5 expression in breast cancers. METHODS: Uptake of 6-[(18)F]FDF was studied in murine EMT-6 and human MCF-7 breast cancer cells over 60 min and compared to [(18)F]FDG. Biodistribution of 6-[(18)F]FDF was determined in BALB/c mice. Tumor uptake was studied with dynamic small animal PET in EMT-6 tumor-bearing BALB/c mice and human xenograft MCF-7 tumor-bearing NIH-III mice in comparison to [(18)F]FDG. 6-[(18)F]FDF metabolism was investigated in mouse blood and urine. RESULTS: 6-[(18)F]FDF is taken up by EMT-6 and MCF-7 breast tumor cells independent of extracellular glucose levels but dependent on the extracellular concentration of fructose. After 60 min, 30±4% (n=9) and 12±1% (n=7) ID/mg protein 6-[(18)F]FDF was found in EMT-6 and MCF-7 cells, respectively. 6-deoxy-6-fluoro-d-fructose had a 10-fold higher potency than fructose to inhibit 6-[(18)F]FDF uptake into EMT-6 cells. Biodistribution in normal mice revealed radioactivity uptake in bone and brain. Radioactivity was accumulated in EMT-6 tumors reaching 3.65±0.30% ID/g (n=3) at 5 min post injection and decreasing to 1.75±0.03% ID/g (n=3) at 120 min post injection. Dynamic small animal PET showed significantly lower radioactivity uptake after 15 min post injection in MCF-7 tumors [standard uptake value (SUV)=0.76±0.05; n=3] compared to EMT-6 tumors (SUV=1.23±0.09; n=3). Interestingly, [(18)F]FDG uptake was significantly different in MCF-7 tumors (SUV(15 min) 0.74±0.12 to SUV(120 min) 0.80±0.15; n=3) versus EMT-6 tumors (SUV(15 min) 1.01±0.33 to SUV(120 min) 1.80±0.25; n=3). 6-[(18)F]FDF was shown to be a substrate for recombinant human ketohexokinase, and it was metabolized rapidly in vivo. CONCLUSION: Based on the GLUT5 specific transport and phosphorylation by ketohexokinase, 6-[(18)F]FDF may represent a novel radiotracer for PET imaging of GLUT5 and ketohexokinase-expressing tumors.

Actions of novel antidiabetic thiazolidinedione, T-174, in animal models of non-insulin-dependent diabetes mellitus (NIDDM) and in cultured muscle cells.

1. The antihyperglycaemic effect and the possible mechanism of action of T-174, a novel thiazolidinedione derivative, was determined in vivo and in vitro. 2. Oral administration of T-174 markedly improved hyperglycaemia, hyperinsulinaemia, hyperlipidaemia, and glucose intolerance in genetically obese and diabetic yellow KK (KK-Ay) mice (0.2-15.5 mg kg(-1) day(-1), for 7 days) and Zucker fatty rats (1.4-11.4 mg kg(-1) day(-1), for 6 days). The ED50 values for the glucose lowering action of T-174 and pioglitazone, another thiazolidinedione antidiabetic, were 1.8 and 29 mg kg(-1) day(-1), respectively in KK-Ay mice; T-174 was about 16 times more potent than pioglitazone. 3. The hypoglycaemic effect of exogenous insulin in KK-Ay mice was enhanced after the administration of T-174. A hyperinsulinaemic euglycaemic clamp study in Zucker fatty rats showed an amelioration of whole-body insulin resistance by the T-174 treatment. 4. Insulin-stimulated glucose metabolism was enhanced in adipocytes from KK-Ay mice treated with T-174. The insulin receptor number of the adipocytes was increased without a change in the affinity of the receptor. 5. The hypomagnesaemia in KK-Ay mice was completely restored by T-174. 6. In cultured L6 myotubes, glucose consumption and [3H]-2-deoxy-glucose transport were enhanced by T-174 (EC50; 6 and 4 microM, respectively). Combination of insulin with T-174 was additive to stimulate glucose disposal. 7. These results suggest that the antihyperglycaemic effect of T-174 was mediated by enhanced insulin action. This was associated with amelioration of the hypomagnesaemia and T-174 directly increased basal and insulin-stimulated glucose utilization by cultured muscle cells.

Kinetic model of 2-deoxyglucose metabolism using brain slices.

A six-compartment, nine-parameter kinetic model of 2-deoxyglucose (2DG) metabolism, which includes bidirectional tissue transport, phosphorylation, two-step dephosphorylation, phosphoisomerization, and conjugation to UDP and macromolecules, has been derived. Data for analysis were obtained from 540- and 1,000-microns-thick hippocampal and hypothalamic brain slices, which were incubated in buffer containing [14C]2DG, frozen, extracted with perchlorate, and separated on anion-exchange columns. Solutions of the equations of the model were fit to the data by means of nonlinear least-squares analysis. These studies suggest that dephosphorylation is adequately described by a single reaction so that the model reduces to eight parameters. The in vitro rate constants for transport, phosphorylation, and dephosphorylation are very similar to prior in vivo results. The phosphoisomerization rate constant is similar to dephosphorylation, so glycosylated macromolecules slowly accumulate and gradually assume larger relative importance as other compounds disappear more rapidly. Rate constants for 540-microns slices from hypothalamus and hippocampus are similar, while 1,000-microns slices have smaller tissue transport constants and larger phosphorylation constants. The rate equation for glucose utilization of this model is relatively insensitive to uncertainties regarding the rate constants. Including later metabolic components in kinetic models improves the calculations of glucose utilization with long isotope exposures.

N-methyl-D-aspartate antagonist reduces stroke size and regional glucose metabolism.

Pharmacological inhibition of cell excitation during focal ischemia was studied in the rat middle cerebral artery occlusion model. The potent and selective N-methyl-D-aspartate antagonist CGS 19755, administered 5 minutes prior to or 5 minutes following permanent middle cerebral artery occlusion, caused a substantial decrease in infarct size, which was associated with reduction of postischemic cerebral glucose hypermetabolism. These data support a role for excitation-induced hypermetabolism in the pathogenesis of infarction following focal cerebrovascular occlusion.

Specific alterations in cardiovascular function and in glucose utilisation within lower brainstem nuclei following intracisternal neuropeptide Y in the conscious rat.

In the conscious normotensive rat, intracisternal neuropeptide Y (NPY) (1.25 nmol i.c.) gave rise to alterations in peripheral haemodynamic variables and glucose use within discrete areas of the CNS as measured by [14C]2-deoxyglucose autoradiography. The haemodynamic response to i.c. NPY comprised a transient hypertension followed by a prolonged hypotension and bradycardia. These cardiovascular responses to NPY were accompanied by a significant reduction in function related glucose use in the area postrema (-29% from vehicle-injected controls), nucleus tractus solitarius (caudal portion -24%, rostral portion -19%), Kolliker-Fuse nucleus (-14%), inferior colliculus (-18%) and subfornical organ (-19%). It is proposed that the area postrema, nucleus tractus solitarius and Kolliker-Fuse nucleus in the brainstem are involved functionally in the haemodynamic response to i.c. NPY.

Regional cerebral glucose metabolism in Turner syndrome.

Regional cerebral glucose metabolism was examined in females with Turner syndrome, a sex chromosome abnormality. Previous studies have found a visual/spatial cognitive anomaly in these women but, to date, no abnormalities in brain structure or function have been associated with the condition. In the present study, decreases in regional metabolism were found in the occipital and parietal cortex. The involvement of the occipital cortex, although consistent with the observed cognitive anomalies, has not been suggested previously as an area dysfunction. Because the occipital cortex is a primary sensory cortex, the reduction of glucose metabolism in the parietal cortex may reflect a lack of innervation from the occipital cortex. Besides insight into the functional specialization of the brain, these findings are also consistent with previous reports on animals regarding the effects of estrogen in brain maturation.

Consequences of chronic phenobarbital treatment on local cerebral glucose utilization in the developing rat.

The influence of a chronic phenobarbital (PhB) treatment on postnatal evolution of local cerebral metabolic rates for glucose (LCMRglc) was studied in 58 cerebral structures of freely moving rats. The animals received a daily subcutaneous injection of PhB at a dose of 50 mg/kg between days 2 and 35 or an equivalent volume of saline for controls and were studied at 5 postnatal stages, i.e. 10, 14, 17, 21 and 35 days, and at the adult stage. Body and brain weights were both reduced by 6-21% over the whole period studied. PhB exposure induced significant decreases in LCMRglc during the period of pharmacological treatment, i.e. until 35 days, except at the stage of 17 days as well as long-term reductions in LCMRglc of adult rats in 36 out of the 58 brain regions studied. These decreases affected all systems studied, sensory systems as well as limbic, hypothalamic, motor and white matter areas. In addition to a growth retardation, PhB also seemed to be able to induce a delay in the acquisition of auditory function which matures early during postnatal life. The long-term deficits in cerebral energy metabolism due to PhB in the adult rat also confirm the behavioral deficits which have been shown previously after early PhB exposure.

Neuronal substrates involved in processing of communicative acoustic signals in tree shrews: a 2-deoxyglucose study.

Autoradiography with [14C]2-deoxyglucose (2-DG) was used to map functional differences in activation of the central auditory pathway in adult tree shrews during presentation of particular acoustic stimuli (low frequency, LFS, and high frequency, HFS, pure sinus tones; social calls, SC). Individuals stimulated with broadband-noise (BBN) were used as controls. Stimulus-specific labelling was found in autoradiographs of cochlear nucleus, superior olivary complex, inferior colliculus and auditory cortex. These findings imply a tonotopic organization at least in these auditory brain areas and indicate differences in the processing of sounds with different functional significance.

Cerebral diaschisis in patients with malignant glioma.

A positron emission tomography study using [18F]-fluorodeoxyglucose was undertaken to identify and quantitate whether diaschisis occurred in cerebral cortex, basal ganglia, or thalamus, as well as in cerebellar cortex and dentate nuclei in patients with malignant glioma. The relationship between diaschisis in these cerebral structures and clinically significant hemiparesis in patients was analysed. A 30% decrease in the regional metabolic rate for glucose in the cerebellar hemisphere contralateral to the tumor, and ipsilateral to the motor deficit, was identified and was statistically significant (p greater than 0.001). Decreased metabolism in the cerebellar hemisphere contralateral to the tumor was not seen in patients without hemiparesis. Parietal lobes affected by tumor had a larger decrease in metabolism than did frontal lobes with tumor (p greater than 0.01). The overall metabolism of the unaffected cerebellar hemisphere, relative to the peak metabolic activity of the brain, was not depressed in patients with tumor. In addition, the activity of subcortical nuclei was relatively unaffected by adjacent tumor or motor deficit.

Cerebral glucose utilization during sleep-wake cycle in man determined by positron emission tomography and [18F]2-fluoro-2-deoxy-D-glucose method.

Using the [18F]fluorodeoxyglucose method and positron emission tomography, we studied cerebral glucose utilization during sleep and wakefulness in 11 young normal subjects. Each of them was studied at least thrice: during wakefulness, slow wave sleep (SWS) and rapid eye movement sleep (REMS), at 1 week intervals. Four stage 3-4 SWS and 4 REMS fulfilled the steady state conditions of the model. The control population consisted of 9 normal age-matched subjects studied twice during wakefulness at, at least, 1 week intervals. Under these conditions, the average difference between the first and the second cerebral glucose metabolic rates (CMRGlu was: -7.91 +/- 15.46%, which does not differ significantly from zero (P = 0.13). During SWS, a significant decrease in CMRGlu was observed as compared to wakefulness (mean difference: -43.80 +/- 14.10%, P less than 0.01). All brain regions were equally affected but thalamic nuclei had significantly lower glucose utilization than the average cortex. During REMS, the CMRGlu were as high as during wakefulness (mean difference: 4.30 +/- 7.40%, P = 0.35). The metabolic pattern during REMS appeared more heterogeneous than at wake. An activation of left temporal and occipital areas is suggested. It is hypothetized that energy requirements for maintaining membrane polarity are reduced during SWS because of a decreased rate of synaptic events. During REMS, cerebral glucose utilization is similar to that of wakefulness, presumably because of reactivated neurotransmission and increased need for ion gradients maintenance.

Maternal thermal stimulation changes metabolic activity in fetal hypothalamus.

To study whether the central nervous system in the perinatal fetal rat can operate during maternal cooling and warming, we examined the 2-deoxy-D-[14C]glucose ([14C]DG) uptake in the fetal brain. Full-term pregnant rats were placed at three different ambient temperatures of 35-37 degrees C, 24-25 degrees C and 0-10 degrees C. Saline containing 20 microCi/100 g of [14C]DG was injected into the superior caval vein in the pregnant rats. Forty-five min after the injection, the mother rats were decapitated and the fetal brains were taken out for autoradiography. The [14C]DG uptake was significantly influenced by maternal thermal stimulation in the hypothalamus and not in other brain regions examined such as the cerebral cortex, the basal ganglia and the limbic nuclei. Glucose utilization in the fetal anterior hypothalamus, paraventricular hypothalamus and dorsomedial hypothalamus significantly increased when the mother rat was exposed to heat compared to when the mother rat was in the thermoneutral condition. During maternal cooling, glucose utilization in the ventromedial hypothalamus and dorsomedial hypothalamus significantly decreased. There was no area activated by cooling and/or inhibited by warming. Compared to a similar study in adult rats (Am. J. Physiol., 248 (1985) R84-92), the present results suggest that although the perinatal fetal brain does not respond to thermal stimulation in terms of glucose utilization as fully as the adults, a few hypothalamic nuclei have already acquired thermal responses, which might be a possible neuronal basis for the thermoregulatory responses just after birth in rats.

The effects of monoamine oxidase inhibition and dopamine uptake blockade on MPTP-induced increase of 2-[3H]deoxyglucose uptake in specific mesencephalic catecholaminergic nuclei.

Intraperitoneal injection in C57 black mice of 30 or 7.5 mg/kg MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) followed 1-2 h later by 0.5 mCi/100 g 2-[3H]deoxyglucose (2-DG) produces an intense increase of 2-DG uptake in the dopaminergic zona compacta (ZC) of the substantia nigra and the adjacent ventral tegmental area (VTA) as seen in autoradiographs. Anesthetized animals also exhibit this reaction. Clorgyline (10 mg/kg), a monoamine oxidase (MAO)-A inhibitor, 30 min before MPTP does not block this reaction. Deprenyl (10 mg/kg), an MAO-B inhibitor, blocks increased 2-DG uptake in ZC and VTA if injected 30 min or 12 h before 7.5 mg/kg MPTP, but has no effect if injected 12 h before 30 mg/kg MPTP. Mazindol (10 mg/kg), a dopamine uptake blocker, is effective in blocking the action of 7.5 mg/kg MPTP, but ineffective against 30 mg/kg MPTP. Heavier doses of MPTP can, in some instances, overcome the actions of MAO inhibitors or dopamine uptake blockers in preventing the increased 2-DG uptake elicited by MPTP. But, essentially, the intensely increased glucose metabolism acutely induced in specific catecholaminergic neurons appears to be another significant pathological feature of the dopaminergic neurotoxicity caused by MPTP which can be prevented by MAO inhibition or dopamine uptake blockade.

Cerebral glucose utilization during diazepam withdrawal in rats.

The diazepam withdrawal syndrome in rats was characterized behaviorally by an increase in spontaneous motor activity, slight body tremor and a lack of convulsions. The 2-deoxyglucose (2-DG) technique was used to measure quantitatively cerebral glucose utilization during diazepam withdrawal and revealed changes in glucose utilization in 30% of the 54 structures evaluated. Areas of increased glucose utilization included medial geniculate, inferior colliculus, visual cortex, mammillary body, dorsal hippocampus, cerebellar flocculus, and zona reticulata and globus pallidus, olfactory cortex, nucleus accumbens and internal capsule. There was no single or consistent relationship between reported benzodiazepine receptor densities and glucose utilization.

Time courses of behavioral and regional cerebral metabolic responses to different doses of meta-chlorophenylpiperazine in awake rats.

The time course and relation to dose of regional cerebral metabolic rates for glucose (rCMRglc) and of motor behavior were measured in awake male adult Fischer-344 rats after administration of meta-chlorophenylpiperazine (MCPP), a serotonin-1B receptor agonist. rCMRglc was determined, using the quantitative autoradiographic [14C]deoxyglucose technique, in 71 brain regions at 5, 15, 30 and 60 min after administration of MCPP 2.5 mg/kg i.p., and at 15 min after MCPP 25 and 40 mg/kg. The time course of performance on a rotating rod was measured periodically for 60 min after MCPP 2.5 mg/kg, a dose which impaired locomotion and reduced rCMRglc maximally at 15-30 min after its administration. At 15 min, rCMRglc declined significantly in 28 (40%) of the areas studied (mean decline 16%). Most regions affected were telencephalic or diencephalic, corresponding to the projection areas of serotonergic fibers arising from the raphe nuclei. After higher doses of MCPP, a behavioral serotonin syndrome was observed with both rCMRglc increases and decreases (25 mg/kg) or only rCMRglc increases (40 mg/kg). Whereas behavioral and metabolic activation induced by high doses of MCPP may result from stimulation at postsynaptic serotonin receptors, rCMRglc reductions and hypomotility produced by MCPP 2.5 mg/kg resemble the effects of serotonin receptor antagonists and suggest that, at this low dose, MCPP acts at modulatory serotonin autoreceptors to reduce endogenous serotonin release.

Effect of propentofylline on the biochemical lesion of the rat brain in aluminium-induced neurotoxicity.

Acute and chronic administration of Al-gluconate (12.7% Al) at the concentration of 1 mg/kg produces edema in the rat brain, as reflected by the increase in water and Na+ content. The permeability for Evans blue is also increased, which indicates the opening of the blood-brain barrier. Higher concentrations of the Al-gluconate (10 mg/kg) change, in acute experiments, the pattern of energy metabolites in the rat brain toward a profile observed in a deep hypoxia. Chronic administration of a low concentration of Al-gluconate (1 mg/kg) increases the local utilization of glucose in 20 of 39 rat brain structures examined. This increase was particularly evident in the structures of the limbic system. Xanthine derivative propentofylline reverses the edema formation in acute and chronic experiments. Hypoxia-like changes in energy metabolism are also reversed by propentofylline. In preliminary experiments propentofylline also suppressed the increased utilization of glucose observed after administration of Al-gluconate. These results suggest that (i) the Al-gluconate model in rats can be used to study Al-neurotoxicity at a very low level of Al, and (ii) the xanthine derivative propentofylline can eventually be used to abolish the Al-neurotoxicity.

Metabolic activation of the fetal rat brain.

In the present study, we tried to examine whether the 2-deoxy-D-[14C]glucose (2-DG) technique is useful to investigate the metabolic activity of fetal rat brain in the maternal uterus. The result shows that metabolic mapping of the fetal brain was clearly obtained by means of the 2-DG technique.

[Quantitative evaluation of myocardial blood flow and myocardial fluoro-deoxyglucose uptake determined by PET before and after aorto-coronary bypass operation in patients with ischemic heart disease].

Absolute myocardial blood flow (MBF) and myocardial FDG uptake (MFU) in the fasting state were determined in 5 patients who underwent aorto-coronary bypass operation, using O-15 water, F-18 fluoro-deoxyglucose and dynamic PET before and after the operation. In the patent graft region, MBF was increased from 0.59 +/- 0.17 ml/min/g to 0.77 +/- 0.14 ml/min/g (p less than 0.05). Mean MBF was increased from 0.69 +/- 0.22 ml/min/g to 0.83 +/- 0.18 ml/min/g (p less than 0.05). MFU in the fasting state was significantly decreased in high MFU region compared with low MFU region (p less than 0.005). Quantitative evaluation of MBF and MFU before aorto-coronary bypass operation was quite useful to determine adequate indication of the operation.

[PET assessment of myocardial viability with glucose loading and fasting FDG images].

Positron emission tomography (PET) offers the potential capability of evaluating tissue viability. We have studied the changes of myocardial F-18 deoxyglucose (FDG) uptake with glucose loading. In a fasting state (for at least 5 hours) and in a glucose loading state (50 g glucose orally one hour before the study) FDG (74-148 MBq) PET studies were performed for 50-60 minutes in 3 cases. 2 were subjects with anterior myocardial infarction (MI) including one with a ventricular aneurysm (case 1), one with a recent MI (case 2). One was a subject of aortic valvular disease without coronary lesions (case 3). Arterial input function (Ca(t)) and myocardial activity (Cm(t)) were derived from the regions of interest (ROI) on the left atrium and from multiple ROI's circumferentially about the myocardium. Net extraction, FU (Fractional Uptake) = Cm(T)/integral of T0 Ca(t)dt, were calculated. Normal segment (N) showed an increase in FU with glucose loading, but the ischemic segment showed no increase (case 1) or a lower increase (case 2) relative to N. This study shows that the increase in FU with glucose loading suggests the persistence of viable myocardium. In conclusion, myocardial viability may be evaluated by comparing FDG images in a glucose loading state with those in a fasting state.

Regional glucose phosphorylation rate in rat brain during acute ethanol intoxication.

The effect of ethanol on regional cerebral metabolic rate for glucose (rCMRglc) was studied in rats using [6-14C]glucose. After intravenous injection, radioactivity was determined in 14 brain regions, corrected for loss of label, and divided by the integral of the arterial plasma glucose concentration measured during tracer circulation. When blood ethanol concentration was maintained at 6 g/l by intravenous infusion of ethanol for approximately 1 h, rCMRglc was found to be reduced significantly in 7 forebrain regions, compared to values of conscious control animals. In 7 further regions including brain stem regions, rCMRglc was not significantly reduced. We conclude that the effects of severe acute alcohol intoxication resemble those of global anesthesia.