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Triple-tracer autoradiography of cerebral blood flow, glucose utilization, and protein synthesis in rat brain.

A triple-tracer autoradiographic technique is described that permits the simultaneous measurement of cerebral blood flow, glucose consumption, and protein synthesis using 131I-iodoantipyrine (131I-IAP), [14C]deoxyglucose ([14C]DG), and 3H-amino acids as radioactive tracers. Autoradiographic differentiation between isotopes was performed by taking advantage of different half-lives, solubility of labeled tracers in a wash solution, and sensitivity of the photographic material to disintegrations of the radionuclides. Blood flow autoradiograms using 131I-IAP were obtained by immediate exposure of brain sections to Kodak NMB film for 24 h. During 131I autoradiography contamination by 3H was absent and by 14C was negligible at tissue concentrations of less than 0.45 microCi/g brain tissue. After complete decay of 131I, reexposure of brain sections to Kodak NMB film for 2 weeks provided autoradiograms that stemmed exclusively from 14C disintegrations without contamination by either 131I or 3H and that represented regional glucose utilization. Brain sections were then wash-incubated for 12 h to remove [14C]DG, [14C]DG-6-phosphate, and free 3H-amino acids from the tissue, and exposed to 3H-sensitive LKB Ultrofilm for 2 weeks for autoradiography of 3H-amino acid incorporation into proteins. 14C radioactivity remaining in the tissue section after wash-incubation was determined by exposing sections again for 2 weeks to Kodak NMB film; the resulting contribution to the blackening of 3H-autoradiograms was corrected for by means of digital subtraction using an image-processing system. The triple-tracer autoradiographic technique was validated in rats under various physiological and pathophysiological conditions. In intact animals extinction correction was necessary only for 3H-autoradiograms. Under pathophysiological conditions, however, significant contamination of 131I by 14C occurred in regions with low blood flow and increased glucose utilization rate; this also required correction by digital subtraction. The interpretation of triple-tracer autoradiographic results is limited by the same restrictions as single-tracer autoradiography, but the simultaneous assessment of the three parameters considerably facilitates the interpretation of the flow/metabolic relationship, particularly under pathological conditions.

Animals↗

Stimulatory effect of endogenous orexin A on gastric emptying and acid secretion independent of gastrin.

Orexin A (OXA) increases food intake and inhibits fasting small bowel motility in rats. The aim of this study was to examine the effect of exogenous OXA and endogenous OXA on gastric emptying, acid secretion, glucose metabolism and distribution of orexin immunoreactivity in the stomach. Rats equipped with a gastric fistula were subjected to intravenous (IV) infusion of OXA or the selective orexin-1 receptor (OX1R) antagonist SB-334867-A during saline or pentagastrin infusion. Gastric emptying was studied with a liquid non-nutrient or nutrient, using 51Cr as radioactive marker. Gastric retention was measured after a 20-min infusion of OXA or SB-334867-A. Plasma concentrations of OXA, insulin, glucagon, glucose and gastrin were studied. Immunohistochemistry against OXA, OX1R and gastrin in gastric tissue was performed. OXA alone had no effect on either acid secretion or gastric emptying. SB-334867-A inhibited both basal and pentagastrin-induced gastric acid secretion and increased gastric retention of the liquid nutrient, but not PEG 4000. Plasma gastrin levels were unchanged by IV OXA or SB-334867-A. Plasma OXA levels decreased after intake of the nutrient meal and infusion of the OX1R antagonist. Only weak effects were seen on plasma glucose and insulin by OXA. Immunoreactivity to OXA and OX1R were found in the mucosa, myenteric cells bodies and varicose nerve fibers in ganglia and circular muscle of the stomach. In conclusion, endogenous OXA influences gastric emptying of a nutrient liquid and gastric acid secretion independent of gastrin. This indicates a role for endogenous OXA, not only in metabolic homeostasis, but also in the pre-absorptive processing of nutrients in the gut.

Animals↗

Steady state models of spore cell metabolism in Dictyostelium discoideum.

Young sorocarps (consisting of a mass of spore cells resting on a stalk) were exposed to low levels of [U-14C]glucose and the spore cells were rapidly separated from stalk cells. Metabolites were isolated from spores and their specific radioactivities compared to these metabolites isolated from the whole organism; i.e. spore plus stalk cells. Based on these data, known reaction rates, and metabolite concentrations, highly constrained steady state models of metabolism in spore and stalk cells were constructed. Direct evidence has been obtained which substantiates earlier predictions regarding cell permeability, the distribution of specific metabolites, and the location of reactions in vivo.

Computers↗

Analysis of activated T cell infiltrates in rat renal allografts by gamma camera imaging after injection of 123iodine-interleukin 2.

Activated T cells bearing receptors for interleukin 2 (IL-2) play an important role in immunity and in immunopathological processes such as allograft rejection. In order to investigate the presence of activated T cells in lymphocytic infiltrates in transplanted kidneys, we investigated the uptake and retention of radioactivity after an intravenous injection of radioiodinated IL-2 in experimentally transplanted rats. IL-2 was enzyme radiolabelled with 123iodine using a glucose oxidase/lactoperoxidase method and shown to retain specific binding on an IL-2 receptor positive cell line, C58E6. To examine the kinetics of 123iodine-interleukin 2 (123I-IL-2) uptake in vivo, animals that had been transplanted five days previously with allogeneic or syngeneic grafts were injected with 123I-IL-2 and then imaged using an external gamma camera. Radioactivity was measured at time points up to 240 min after intravenous injection of 123I-IL-2. Four groups of animals were examined: allogeneic grafts (n = 7); syngeneic grafts (n = 6); ischaemic native kidneys (n = 5) all following injection with 123I-IL-2; and allogeneic transplants (n = 5) after injection of 123I-lactalbumin, an irrelevant molecule of similar molecular weight to IL-2. The peak radioactivity after injection was measured and the amount of radioactivity retained in the graft at increasing time intervals after injection was expressed as a function of initial peak radioactivity. At four hours after injection of 123I-IL-2, mean retention of activity by rejecting grafts was 77(+/- 2.68)% of peak activity, compared to 45(+/- 6.38)% in syngeneic controls (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Sites of glycogen synthesis in rat liver cells as shown by electron microscope radioautography after administration of glucose-H3.

Glycogen synthesis was investigated by giving tritium (H(3))-labeled glucose with carrier to fasted rats in vivo or incubating liver slices from fasted rats in vitro using a glucose-H(3)-containing medium. After 15 min or 1 hr, pieces of liver were fixed and radioautographed for light and electron microscopy. In vivo and in vitro, radioautographic reactions appeared over "glycogen areas" and over zones transitional between these areas and ergastoplasm. Treatment of sections by alpha amylase removed all but about 5% of the radioactivity, so that about 95% of it consisted of glycogen (synthesized during the 15 min or 1 hr elapsing after administration of glucose-H(3)). Within glycogen areas and transitional zones, most silver grains were over or very close to glycogen granules and smooth (or partly smooth) vesicles. Presumably, much of the label was added onto growing glycogen granules, in accord with the biochemical view that glycogen may serve as substrate for further glycogen synthesis. The few silver grains located far from glycogen granules-15% at the 15 min interval in vivo-approximated smooth (or partly smooth) vesicles of endoplasmic reticulum. This observation raised the possibility that smooth membranes play a role in glucose uptake at an early stage in de novo formation of glycogen granules.

Amylases↗

Rapid feasibility studies of tracers for positron emission tomography: high-resolution PET in small animals with kinetic analysis.

The development of methods for production of a radiotracer for use in human studies with positron emission tomography (PET) is often a time-consuming process of optimizing radiolabelling yields and handling procedures. Sometimes the radiotracer is not the original drug, but rather a derivative with unknown in vivo pharmacological properties. We have developed a fast and simple method of testing putative new PET tracers in vivo in small animals. The procedure has been validated in rats with different PET tracers with known kinetic and pharmacological properties ([2-18F]2-fluoro-2-deoxy-D-glucose, [N-methyl-11C]Ro 15-1788, and [15O]butanol). The tracer concentration in arterial blood was continuously measured to obtain the brain input function. Following image reconstruction of the scans, time-activity curves of selected regions of interest were generated. Estimations of CMRglc (1.0 +/- 0.2 mumol g-1 min-1), CBF (1.4 +/- 0.4 ml g-1 min-1) and transport rate constants for [N-methyl-11C]Ro 15-1788 (K1 = 0.44 +/- 0.01 ml g-1 min-1 and k2 = 0.099 +/- 0.005 min-1) as well as calculated first pass extraction (0.32 +/- 0.1) are in reasonable agreement with literature values. Small animal studies require minimal amounts of radioactivity and can be performed without sterility and toxicology tests. They may serve as a preliminary basis for radiation safety calculations because whole body scans can be performed even with a head scanner.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Effects of insulin in vitro on protein turnover in rat epitrochlearis muscle.

Rates of protein synthesis and degradation were measured in the isolated rat epitrochlearis muscle by radiotracer techniques, by using the specific radioactivity of tRNA-bound amino acid as precursor for protein synthesis. The tissue maintained linear rates of protein synthesis for 3 h of incubation in the presence of amino acids and glucose and in the absence of insulin. Under these conditions, however, the muscles were in negative nitrogen balance, with rates of protein degradation exceeding rates of protein synthesis. Under steady-state conditions of labelling, the specific radioactivities of tRNA-bound leucine, phenylalanine and valine were significantly less than their respective values in the incubation medium, at concentrations in the medium varying from 1 to 10 times those in normal rat serum. Insulin caused a dose- and time-dependent increase in tRNA-based protein synthesis rates, more than doubling rates at 5 and 50 ng of insulin/ml. At the lower, physiological, concentration of insulin, the stimulation of protein synthesis was not observed until the third hour of incubation with the hormone, whereas the rate of protein synthesis at the higher concentration was elevated during the second hour. There were no delays in the stimulation by insulin of glucose conversion into glycogen. The delayed stimulatory effects of insulin on the rate of protein synthesis brought the tissue to a nitrogen balance near zero. The presence of the hormone also prevented the increase in the rate of protein degradation seen in the third hour of incubation in the absence of the hormone. These studies demonstrate the viability of the incubated rat epitrochlearis muscle with respect to protein metabolism and sensitivity to the protein anabolic effects of physiological concentrations of insulin, and indicate that the preparation is a suitable experimental model for the study of the control of protein metabolism in fast-twitch skeletal muscle.

Amino Acids↗

Alpha 1-adrenergic regulation of ketogenesis in isolated rat hepatocytes.

Studies were conducted to clarify the effects of noradrenaline on oleate metabolism in isolated hepatocytes from fed rats. Noradrenaline caused an inhibition of ketogenesis from oleate along with a stimulation of glucose release through alpha 1-adrenergic receptors. Anti-ketogenic action of noradrenaline was confirmed by the suppression of the formation of radioactive acid-soluble products from [1-14C]oleate in response to this agent. Noradrenaline increased the conversion of [1-14C]oleate into 14CO2 but failed to affect [1-14C]oleate esterification. When hepatocytes were incubated in a medium containing 1 mM EGTA but no Ca2+, the effects of noradrenaline on oleate oxidation were negated. On the other hand, noradrenaline-induced increase in glucose release remained unchanged even in the absence of Ca2+ in the incubation medium. Decrease in ketogenesis and increase in glucose release produced by vasopressin was completely abolished by calcium depletion. Noradrenaline caused a significant increase in cAMP levels in both the presence and absence of Ca2+, although the effect was more marked in the latter. Vasopressin did not affect it. The noradrenaline-induced increase in cAMP and glucose release in the absence of Ca2+ was also mediated by alpha 1-adrenergic receptors. These data are discussed and it is suggested that alpha 1-adrenergic agonists may control hepatic ketogenesis and glycogenolysis through two separate signal transduction mechanisms, i.e., a calcium-mobilizing system which is common with vasopressin, and a cAMP generation system which vasopressin lacks.

Animals↗

Influence of dietary composition on gluconeogenesis from L-(U-14C)glutamate in rainbow trout (Salmo gairdneri).

The effect of dietary composition (high-protein, high-carbohydrate and high-fat diets) and starvation on in totum gluconeogenesis from L-(U-14C)glutamate was studied in the rainbow trout. High-fat and high-carbohydrate diets produced a significant hyperglycaemia. Lower blood glucose values were obtained in trout fed on a high-protein diet. Liver glycogen levels were significantly lower in trout fed on carbohydrate-free diets (high-protein and high-fat diets) and in starved fish. Gluconeogenesis from L-(U-14C)glutamate was markedly reduced in fish given the high-carbohydrate diet and significantly enhanced in starved fish. Radioactive liver glycogen was higher in starved fish, although the amount of radioactivity incorporated into glycogen was very low.

Animals↗

Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high-glucose medium.

Nodular expansion of glomerular mesangium with increased amounts of extracellular matrix (ECM) material is pathognomic of diabetic nephropathy. The precise mechanisms involved in this accumulation are unknown. Recently, we reported using a solid-phase enzyme-linked immunosorbent assay (ELISA) technique that glomerular mesangial cells, the principal cell type residing in glomerular mesangium, accumulate 50-60% more fibronectin (FN), laminin (LM), and type IV collagen (T-IV) when cultured in medium containing high glucose (30 mM) (S. H. Ayo, R. A. Rodnik, J. Garoni, W. F. Glass II, and J. I. Kreiberg. Am. J. Pathol. 136: 1339-1348, 1990). ECM assembly is controlled by its rate of synthesis and degradation, as well as its binding and rate of incorporation into the ECM. To elucidate the mechanisms involved, pulse-chase experiments were designed to estimate ECM protein synthesis from the incorporation of Trans-35S [( 35S]methionine, [35S]cysteine) into immunoprecipitated FN, LM, and T-IV. mRNA levels were examined, and degradation rates were estimated from the disappearance of radioactivity from matrix proteins in mesangial cells previously incubated with Trans-35S. One week of growth in 30 mM glucose resulted in approximately 40-50% increase in the synthesis of all three matrix proteins compared with 10 mM glucose-grown cells. This was accompanied by a significant increase in the transcripts for all three matrix proteins (approximately twofold). The specific activity of the radiolabel in trichloroacetic acid-precipitable cell protein showed no difference between cells grown in 10 or 30 mM glucose, indicating that total protein synthesis was unchanged. After 1 wk, the rate of FN, LM, and T-IV collagen degradation was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Cyclic beta-(1,2)-glucan synthesis in Rhizobiaceae: roles of the 319-kilodalton protein intermediate.

Cyclic beta-(1,2)-glucans are synthesized by members of the Rhizobiaceae family through protein-linked oligosaccharides as intermediates. The protein moiety is a large inner membrane molecule of about 319 kDa. In Agrobacterium tumefaciens and in Rhizobium meliloti the protein is termed ChvB and NdvB, respectively. Inner membranes of R. meliloti 102F34 and A. tumefaciens A348 were first incubated with UDP-[14C]Glc and then solubilized with Triton X-100 and analyzed by polyacrylamide gel electrophoresis under native conditions. A radioactive band corresponding to the 319-kDa protein was detected in both bacteria. Triton-solubilized inner membranes of A. tumefaciens were submitted to native electrophoresis and then assayed for oligosaccharide-protein intermediate formation in situ by incubating the gel with UDP-[14C]Glc. A [14C]glucose-labeled protein with an electrophoretic mobility identical to that corresponding to the 319-kDa [14C]glucan protein intermediate was detected. In addition, protein-linked radioactivity was partially chased when the gel was incubated with unlabeled UDP-Glc. A heterogeneous family of cyclic beta-(1,2)-glucans was formed upon incubation of the gel portion containing the 319-kDa protein intermediate with UDP-[14C]Glc. A protein with an electrophoretic behavior similar to the 319-kDa protein intermediate was "in gel" labeled by using Triton-solubilized inner membranes of an A. tumefaciens exoC mutant, which contains a protein intermediate without nascent glucan. These results indicate that initiation (protein glucosylation), elongation, and cyclization were catalyzed in situ. Therefore, the three enzymatic activities detected in situ reside in a unique protein component (i.e., cyclic beta-(1,2)-glucan synthase). It is suggested that the protein component is the 319-kDa protein intermediate, which might catalyze the overall cyclic beta-(1,2)-glucan synthesis.

Bacterial Proteins↗

NMDA-receptor activity visualized with (S)-[N-methyl-11C]ketamine and positron emission tomography in patients with medial temporal lobe epilepsy.

PURPOSE: To determine whether neurochemical activation of the N-methyl-D-aspartate (NMDA) receptor-gated ion channel shows quantitative changes, measured as binding of 11C-labeled (S)-[N-methyl]ketamine, in patients with medial temporal lobe epilepsy (MTLE). METHODS: Eight patients with MTLE who were evaluated regarding epilepsy surgery underwent positron emission tomography (PET) with (S)-[N-methyl-11C]ketamine. The presurgical investigations included magnetic resonance imaging (MRI), PET with 18F-fluoro-deoxyglucose (18FDG), and seizure monitoring by using video-EEG. The uptake of (S)-[N-methyl-11C]ketamine in the temporal lobe of ictal onset was compared with the contralateral side and correlated to changes in regional glucose metabolism measured by PET with 18FDG. RESULTS: (S)-[N-methyl-11C]ketamine rapidly reached the brain, and high radioactivities were measured in the striatum, thalamic nuclei, and cortical regions. Overall the brain uptake and regional binding potentials of (S)-[N-methyl-11C]ketamine were similar to measurements observed previously in healthy controls. However, 20 min after administration, when blood flow influence was negligible, a side-to-side comparison revealed a 9-34% reduction of tracer radioactivity in the temporal lobes of ictal onset. At earlier times, the differences in binding potentials were less pronounced, 9-21%. The magnitude and distribution of the reduction were similar to the metabolic pattern seen on PET scans with 18FDG. CONCLUSIONS: Radioactivity uptake of intravenously administered (S)-[N-methyl-11C]ketamine was reduced in temporal lobes of ictal in patients with TLE. This may reflect reduced NMDA-receptor density, reduced perfusion, focal atrophy, or other factors.

Adult↗

Ocular blood flow and retinal metabolism in abyssinian cats with hereditary retinal degeneration.

PURPOSE: To investigate if retinal blood flow decreases with progression of the disease in Abyssinian cats with progressive retinal atrophy (PRA), to examine if the choroidal blood flow was affected by the disease, and to determine the uptake of glucose and formation of lactate in the outer retina. METHODS: Local blood flow in different parts of the eye was determined with radioactive microspheres, in 9 normal cats and in 10 cats at different stages of PRA. Three blood flow determinations were made in each animal, during control conditions, after IV administration of indomethacin and after subsequent administration of N(omega)-nitro-L-arginine (L-NA). Blood samples from a choroidal vein and a femoral artery were collected to determine the retinal formation of lactate and uptake of glucose. RESULTS: In Abyssinian cats with PRA (n = 10), the retinal blood flow was significantly (P < or = 0.01) lower than in normal cats (n = 9) during control conditions, 6.4 +/- 1.7 compared with 14.1 +/- 1.9 g min(-1) x (100 g)(-1). The vascular resistance in the iris and ciliary body was significantly higher in the cats at a late stage of PRA, both compared with normal cats and to cats at an early stage of the disease, whereas the choroidal vascular resistance was not significantly affected. Indomethacin had no effect on ocular blood flows in normal cats, but in cats with PRA, iridal blood flow was more than doubled after indomethacin. The retinal formation of lactate was significantly (P < or = 0.001) lower in cats with PRA than in normal cats, 0.111 +/- 0.035 (n = 8) compared with 0.318 +/- 0.024 (n = 8) micromol x min(-1). The uptake of glucose was not significantly different in cats with PRA. CONCLUSIONS: Retinal blood flow is severely decreased in Abyssinian cats at a late stage of retinal degeneration, whereas the choroidal microcirculation is not significantly affected by the disease. At a late stage of retinal degeneration, vascular resistance in the iris is significantly increased, which at least in part could be caused by cyxlooxygenase products.

Animals↗

In vivo assessment of glucose metabolism in hepatocellular carcinoma with FDG-PET.

UNLABELLED: The present study was designed to assess glucose metabolism in hepatocellular carcinoma (HCC) with PET and [18F]fluorodeoxyglucose (FDG) and to compare the results with the measured in vitro enzymatic activity of glucose metabolism and the histologic grading of HCC. METHODS: Dynamic FDG-PET scans were obtained in 17 preoperative patients with HCC. From the serial tissue and arterial radioactivities obtained by dynamic PET, FDG kinetic rate constants (K1 to k4) were obtained. The standardized uptake value (SUV) was also determined from the images acquired 48 to 60 min after FDG administration. These PET results were compared with hexokinase and glucose-6-phosphatase (G6Pase) activities and histologic grading of HCC in surgically resected tumor materials. According to histologic grading, the tumors were divided into low-grade and high-grade HCCs. RESULTS: The k3 and SUV of high-grade HCCs were significantly higher than those of low-grade HCCs (p < 0.005, each). In addition, high correlations were observed between the hexokinase activities and these two parameters (r = 0.715 0.768, respectively). In some HCCs, relatively high G6Pase activities and k4 values modified tumor FDG uptake. CONCLUSION: FDG PET is a valuable method for assessing glucose metabolism and histologic grading of HCC.

Carcinoma, Hepatocellular↗

Carminic acid, a non-competitive inhibitor of kidney UDP-glucose:galactosylhydroxylysine-collagen glucosyltransferase.

1. UDP-glucose:galactosylhydroxylsine-collagen glucosyltransferase was purified 12-fold from rat kidney. 2. An assay using calf-skin gelatin as substrate showed time- and enzyme-dependent incorporation; KmS for UDP-glucose and gelatin were 16-7 microM and 4.5 mg/ml, respectively. 3. Column chromatography of the alkaline hydrolysate of reaction product on Dowex 50W-4X(H+) showed that 84% of the radioactivity was in the glycosylgalactosylhydroxylsine peak. 4. Carminic acid inhibited collagen glycosyltransferase; a dose-dependent study showed a two-stage inhibition and kinetic analysis by double-reciprocal plots at varying UDP-glucose concentrations revealed a non-competitive mode of inhibition.

Animals↗