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PubMed1977

Release of immunoreactive and radioactively prelabelled endogenous (pro)-insulin from isolated islets of rat pancreas in the presence of exogenous insulin.

To study the influence of insulin on its own secretion, collagenase-isolated islets of rat pancreas were prelabelled with [3H]leucine for 2 h. After washing the islets, (pro-)insulin release was stimulated by glucose in the presence or absence of exogenous insulin (up to 2-5 mu./ml). Hormone release was unchanged by the presence of exogenous insulin as judged by determination of both immunoreactive insulin and radioactivity incorporated into the proinsulin and insulin fractions of the medium. No direct feedback mechanism for insulin secretion was apparent from this study.

Animals↗
PubMed1975

Dissociation by cytochalasin B of movement, DNA synthesis and transport in 3T3 cells.

Cytochalasin B was used as a tool to study the inter-relationships between cell movement, the reinitiated DNA synthesis and the enhanced transport of specific small molecules stimulated by serum in quiescent 3T3 cells. Cytochalasin at concentrations of less than 1 mug/ml inhibits serum-stimulated movement within the monolayer and migration into a wound. Even at ten times this concentration there is little effect on the increase in DNA in the culture, indicating that movement away from neighboring cells is not required for the initiation of DNA synthesis. While DNA synthesis is not inhibited by concentrations of cytochalasin up to 10 mug/ml, the increased thymidine transport which is associated with the onset of the S phase of the cell cycle is inhibited and DNA synthesis cannot be measured by the labelling of nuclei with radioactive thymidine. Cytochalasin has a differential effect on the early transport changes produced by serum addition. Glucose transport is inhibited by low concentrations of the drug (less than 1 mug/ml) while the enhanced uptake of phosphate and uridine is unaffected by a 10-fold increase in concentration. Although the doses of cytochalasin required for 50% inhibition of hexose uptake and of cell movement are the same, no causal relationship between sugar transport and locomotion can be demonstrated. Cytochalasin affects membrane functions in at least two different ways. The drug inhibits the uptake of glucose directly but affects only the S-phase associated increase in thymidine transport.

Animals↗
PubMed1997

Increased glucagon action on lactate gluconeogenesis in perfused liver of dexamethasone-treated rats.

To clearly understand the hyperglycemic action of glucocorticoids, we studied the action of glucagon on lactate gluconeogenesis in the liver of rats 7 days after adrenalectomy and after treatment with 1 mg/kg dexamethasone for 7 days. The liver was isolated and cyclically perfused at 20 ml/min with 25 ml of perfusion medium containing 5 mM lactate, [U-14C]lactate, and 0-100 ng/ml glucagon. In the absence of glucagon, incorporation of [14C]lactate into glucose carbon 1 did not change significantly in the adrenalectomized rat liver (1.66 +/- 0.12% of total radioactivity for 5 min) and increased in the dexamethasone-treated rat liver (3. 61 +/- 0.54%, P < 0.01) compared to the normal rat liver (1.99 +/- 0. 28%). The response of lactate gluconeogenesis to glucagon was extremely blunted in the adrenalectomized rat liver and was much larger in the dexamethasone-treated rat than in the normal rat liver (at a glucagon concentration of 100 ng/ml, 2.13 +/- 0.33, 8.55 +/- 1. 06, and 4.61 +/- 0.53% for 5 min, respectively). Glucagon binding to liver plasma membrane was not changed by adrenalectomy and was decreased by dexamethasone treatment. These results suggest that glucocorticoids induce hyperglycemia by increasing the response to glucagon, together with the high basal activity of hepatic gluconeogenesis. In addition, these effects do not occur through changes in glucagon binding to receptors.

Animals↗
PubMed1994

Effects of aprotinin on acute recovery of cerebral metabolism in piglets after hypothermic circulatory arrest.

Brain protection during cardiopulmonary bypass and hypothermic circulatory arrest is incomplete. Activation of blood protease cascades may contribute to cellular injury under these conditions. To test this hypothesis, effects of the protease inhibitor aprotinin on recovery of brain energy metabolism after hypothermic circulatory arrest were studied in the piglet. Twenty-four 4-week-old piglets (10 aprotinin-treated and 14 control) underwent core cooling, 1 hour of circulatory arrest at 15 degrees C, reperfusion and rewarming (45 minutes), and normothermic perfusion (3 hours) on cardiopulmonary bypass. Cerebral high-energy phosphate concentration and intracellular pH were studied by phosphorus-31 magnetic resonance spectroscopy in 12 animals. In the remaining animals cerebral and regional blood flow were measured with radioactive microspheres and carotid artery blood flow was measured with an electromagnetic flowmeter. Cerebral oxygen and glucose extraction were measured, and vascular resistance responses to endothelium-dependent (acetylcholine) and -independent (nitroglycerin) vasodilators were calculated. Recovery of cerebral adenosine triphosphate (p = 0.02) and intracellular pH (p = 0.04) in the initial 30 minutes of reperfusion was accelerated in the aprotinin-treated piglets. These piglets showed a greater in vivo cerebral and systemic endothelium-mediated vasodilation (acetylcholine response: cerebral p < 0.01, systemic p = 0.04) after reperfusion. The response to endothelium-independent vasodilation (nitroglycerin) was the same in both groups. Carotid blood flow tended to be greater at 20 minutes of reperfusion and less during 45 to 80 minutes after reperfusion in the aprotinin-treated animals. Brain water content postoperatively was 0.8077 in the aprotinin group and 0.8122 in control animals (p = 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

ATPase activities in peroxisome-proliferating yeast.

Preliminary studies on yeast peroxisomes have suggested that the membrane of these organelles may contain a proton-pumping ATPase. It has been reported that peroxisome-associated activity is similar to the F0-F1 mitochondrial type ATPase in its sensitivity to azide at pH 9.0, but characteristics of the plasma membrane type ATPase are also evident in peroxisomal preparations in that they exhibit pH 6.5 activity that is sensitive to vanadate. A comparative study of the prominent organellar ATPase activities was undertaken as a probe into the existence of an enzyme that is unique to the peroxisome, and biochemical properties of yeast mitochondrial, plasma membrane, together with peroxisomally-associated H(+)-ATPases are presented. Enzyme marker analysis of sucrose gradient fractions revealed a high degree of correlation between the amount of azide-sensitive pH 9.0 ATPase activity and that of the mitochondrial membrane marker, cytochrome c oxidase, in peroxisomal preparations. Purified mitochondrial and peroxisomally-associated activities were highly sensitive to the presence of sodium azide, N,N' -dicyclohexylcarbodiimide (DCCD) and venturicidin when measured at pH 9.0. Comparisons of peroxisomal activities with those of the purified plasma membrane at pH 6.0 in the presence of azide showed similar sensitivity profiles with respect to inhibitors of yeast plasma membrane ATPases such as vanadate and p-chloromercuriphenyl-sulfonic acid (CMP). Purified peroxisomal membranes, furthermore, reacted with antibody to the mitochondrial F1 subunit (as revealed by Western blot analysis), and [35S] methionine-labeled, glucose-grown cells processed with unlabeled methanol-grown cells, yielded sucrose gradient fractions that were radioactive in bands that were also recognized by F1 antibody. Isolated fractions in these experiments had similar ratios of cpm:pH 9.0 ATPase activities, suggesting that this activity is mitochondrial in origin. The data presented for the characteristics of the peroxisomally-associated activity strongly suggest that the majority of the ATPase activity found in peroxisomal preparations is derived from other organelles.

Adenosine Triphosphatases↗
PubMed1983

Extent of 14C incorporation into cholesterol of infant skin fibroblasts incubated with carboxyl-labeled oleic, -linoleic or -arachidonic acid.

In order to determine the incorporation of C1-14C derived from mono- and poly-unsaturated fatty acids into cholesterol of human cells cultured in exponential phase, infant skin fibroblasts (SF) were used at the 5th passage. On Day 6, the SF were preincubated 36 h in a medium containing 5 per cent lipoprotein-deficient serum, and thereafter [1-14C] oleic, -linoleic or -arachidonic acid-without (OL1, LI1 and AR1 group SF), or with the addition of 0.25 mM cold fatty acids (OL2), LI2 and AR2 group SF). Cholesterol specific radioactivity (SRA) peaked 1 h after, and leveled off afterwards in the OL1, LI1 and AR1 groups. Cholesterol-SRA was relatively low in the other groups, but increased progressively, giving a biphasic response: C1-14C derived from from linoleic and arachidonic acids was actively incorporated into cholesterol during the first hours, as compared to C1-14C derived from oleic acid, but stabilized between 6 and 12 h for the LI2 and AR2 group SF incubation. This result appears to be due to the stimulation of pyruvate decarboxylation, observed elsewhere, and consequently to the dilution of the radioactive units in a large pool of non-labeled acetyl-CoA units derived from glucose, when these SF were incubated with 0.25 mM polyunsaturated fatty acids.

Arachidonic Acid↗
PubMed1967

Translocation of C in the sugarcane plant during the day and night.

The time-course of translocation of (14)C from the blades of the sugarcane plant was investigated by analysis and radioactive counting of successive samples punched from a single blade. In 1 experiment, the time-course was studied by determining the specific activity of the carbon dioxide respired by the roots.The rate of translocation, expressed as percentage, was highest immediately after the application of the radioactive carbon dioxide. Morning-made photosynthate translocated a higher percentage during the morning than during the afternoon in 90-minute periods in the light. Afternoon-made photosynthate translocated as well or better than morning-made photosynthate for the first hour in the light.The leaf-disk data and the specific activity of the carbon dioxide respired by the roots corresponded by showing lower rates of translocation by night than by day for several successive days. Also, the translocation of (12)C sucrose was slower at night.The (14)C sucrose translocated by day was made primarily by photosynthesis; the sucrose translocated by night was made primarily by the conversion of other labeled compounds, e.g. organic acids, organic phosphates, and insoluble residue.The radioactive constituent of the residue, which was converted to sucrose, was tentatively identified as a glucose-xylose-glucuronic acid hemicellulose, with most or all of the (14)C in the glucose moiety.Translocation of sucrose may be triggered by different mechanisms during the night than the day. The conversion of insoluble residue to sucrose by increasing the osmotic potential at the source would favor a pressure-flow mechanism for nocturnal translocation; whereas translocation by day is thought to be a process of phototranslocation, a photoactivation of the translocation mechanism.

Journal Article↗
PubMed1974

Sugar metabolism in transketolase mutants of Escherichia coli.

This paper continues the description of transketolase mutants of Escherichia coli; they are absolutely unable to grow on pentoses, but slightly "leaky" with respect to their aromatic requirement (B. L. Josephson and D. G. Fraenkel, 1969). Several experiments have explored the degree of leakiness and shown it to be low. There is little conversion of radioactive xylose to carbon dioxide. The labeling of ribose in cells grown on [1-(14)C]glucose and [2-(14)C]glucose accords with its origin being chiefly by the oxidative pathway. A mutant lacking both transketolase and gluconate-6-phosphate dehydrogenase has been constructed; it requires supplementation with pentose. Pentoses are inhibitory to growth of transketolase mutants, but high levels of pentose phosphates do not accumulate in this situation. Several experimental results are suggestive of regulation of metabolic flow in the oxidative branch of the hexose monophosphate shunt.

Aerobiosis↗
PubMed2004

Endogenous glucose production in type 2 diabetes: basal and postprandial. Role of diurnal rhythms.

Glycemia in type 2 diabetes is characterized by a nonsteady but stable diurnal cycle. This leads to morning fasting hyperglycemia. It arises from an underlying circadian pattern in endogenous glucose production because the metabolic clearance rate of glucose is decreased but constant. Therefore, it is important to use appropriate nonsteady tracer methods to measure this rate even under basal conditions. Postprandially, in diabetes, the endogenous glucose production continues to decrease, with only minor deviations from the slope of the basal curve. This suggests a decoupling of endogenous glucose production from the regulatory factors (insulin, glucose) that prevail under normal circumstances. As the duration of diabetes increases, metabolic clearance of glucose continues to deteriorate. This may be partially compensated by a decrease in glucose production. This rate remains, however, inappropriate because its impact on glycemia does not decline.

Circadian Rhythm↗
PubMed1981

Estrous behavior and pituitary and brain cell nuclear retention of [3H]estradiol in chronically insulin-deficient female rats.

Chronic insulin deficiency, produced by administration of 60 mg/kg streptozotocin, leads to reduced estrous behavior and lower estradiol uptake in both whole homogenate and cell nuclear fractions of hypothalamus-preoptic area and pituitary gland in ovariectomized rats. High dietary fat intake produces an ameliorative effect on both lordosis behavior and estradiol uptake, relative to animals fed a standard, carbohydrate-rich diet. Plasma glucose levels are significantly correlated with estradiol uptake levels in brain and pituitary, and with plasma radioactivity levels. The data support the hypothesis that some of the reproductive dysfunctions manifest in female diabetic rats are the result of alterations in nuclear binding of estradiol by central nervous tissue, and that chronic fuel deprivation may represent an important correlate of disruptions in normal steroid action among diabetics.

Animals↗
PubMedDate not supplied

A new method for the identification of cerebrospinal fluid leakage.

Apart from the use of anamnestic data the most common techniques for the diagnosis of CSF leakage have included X-ray studies, and chemical analysis of glucose, protein, and electrolytes of the fluid obtained from the nose or ear, intrathecal staining, and radioactive cisternography. These studies, although useful, have not always succeeded in demonstrating the CSF leakage, especially when the leak is delayed, small, or contaminated. A new immunochemical technique for the identification of the CSF leakage is described. It is based on demonstration of an extra band of transferrin located in the beta 2-fraction of protein electrophoresis of CSF. This beta 2-transferrin is pathognomonic for liquor and could not be demonstrated in serum, nasal secretions, saliva, tears, or peri- and endolymph. After routine protein electrophoresis on cellulose acetate membranes, the transferrins are identified by application of anti-transferrin on beta-regions. Stained precipitates in both beta-regions demonstrate clearly the presence of CSF. Compared with other methods, the new technique offers many advantages. The amount of sample needed for the procedure is small, moderate contamination does not invalidate it, it makes it possible to localize the leak by differential suction, and it is absolutely safe for the patient.

Blood Protein Electrophoresis↗
PubMed1989

Studies on prolonged acute regional ischemia. V. Metabolic support of remote myocardium during left ventricular power failure.

This study tests the hypothesis that metabolic support of remote "nonischemic" myocardium during acute infarction will reverse the trend toward cardiogenic shock. Thirty-seven dogs underwent ligation of the left anterior descending coronary artery and 50% stenosis of the circumflex coronary artery. Irreversible ventricular fibrillation developed in 11 of them. The 26 survivors were observed for up to 6 hours; global and regional left ventricular function (cardiac index, stroke work index, ultrasonic crystals) and regional blood flow (radioactive microspheres) were measured. After 2 hours, eight dogs received an intravenous infusion of glutamate/aspartate, glucose-insulin-potassium, coenzyme Q10, and 2-mercapto-propionyl-glycine for 4 hours. Five dogs received the mannitol infusion to raise serum osmolarity 30 mOsm. Four additional dogs received the intravenous substrate infusions over 4 hours without undergoing ischemia. The substrate infusion for 4 hours caused no change in regional or global cardiac function in the four control dogs. Three of nine untreated dogs died of cardiogenic shock, and progressive left ventricular power failure occurred in the six others (40% decrease in cardiac index, 50% decrease in stroke work index, p less than 0.05) because of persistent dyskinesia in the left anterior descending region (-40% of systolic shortening, p less than 0.05) and hypocontractility in the circumflex region (48% of control systolic shortening, p less than 0.05), despite normal transmural blood flow in the posterior left ventricular wall (76 ml/100 gm/min). In contrast, in treated dogs, hypercontractility recovered in the circumflex segment (138% of systolic shortening) and stroke work index rose to control levels (91%) without a change in regional blood flow. Mannitol infusion did not improve hemodynamics or avoid the development of progressive left ventricular power failure. We conclude that cardiogenic shock after myocardial infarction is due, in large part, to impaired ability of "nonischemic" myocardium to maintain hypercontractility. This limitation can be prevented by metabolic support of viable muscle, and the data imply that intravenous substrate infusions may be helpful before definitive treatment (i.e., coronary artery bypass grafting) is undertaken.

Animals↗
PubMed1987

Topography of capillary density, glucose metabolism, and microvascular function within the rat inferior colliculus.

A midbrain nucleus of the auditory system, the inferior colliculus, was used as a model for analyzing spatial correlations or "coupling" among capillary density, tissue glucose metabolism, and several measures of microvascular function in the rat. The capillary bed of the inferior colliculus was examined with stereological techniques, and physiological measures were obtained with radioactive tracers, quantitative autoradiography, and image processing. Within the colliculus, capillary density, volume fraction, length, and surface area were highest in the central nucleus where the packing densities of neuropil and perikarya are greatest. Rates of glucose metabolism and blood flow correlated closely with capillary density in a 3 X 2 matrix of collicular subregions in the sagittal and coronal planes. The strength of this correlation suggests that estimates of capillary density can be made from measurements of tissue glucose metabolism within this structure under normal conditions. Microvascular blood volume and transcapillary flux of a neutral amino acid, alpha-aminoisobutyric acid, were homogeneous throughout the colliculus. The studies demonstrate quantitatively in a single brain nucleus a close correspondence between cytoarchitecture, richness of the capillary bed, and complexity of neural activity (inferred from local measures of glucose metabolism and blood flow). Such relationships were suggested by Craigie 67 years ago.

Animals↗
PubMed1979

Effects of ethynyloestradiol on the metabolism of [1-14C]oleate by perfused livers and hepatocytes from female rats.

Normal female rats were given 15mug of ethynyloestradiol/kg body wt. for 14 days and were killed on day 15 after starvation for 12-14h. The livers were isolated and were perfused with a medium containing washed bovine erythrocytes, bovine serum albumin, glucose and [1-(14)C]oleic acid; 414mumol of oleate were infused/h during a 3h experimental period. The output of bile and the flow of perfusate/g of liver were decreased in livers from animals pretreated with ethynyloestradiol, whereas the liver weight was increased slightly. The rates of uptake and of utilization of [1-(14)C]oleate were measured when the concentration of unesterified fatty acid in the perfusate plasma was constant. The uptake of unesterified fatty acid was unaffected by pretreatment of the animal with oestrogen; however, the rate of incorporation of [1-(14)C]oleate into hepatic and perfusate triacylglycerol was stimulated, whereas the rate of conversion into ketone bodies was impaired by treatment of the rat with ethynyloestradiol. Pretreatment of the rat with ethynyloestradiol increased the output of very-low-density lipoprotein triacylglycerol, cholesterol, phospholipid and protein. The production of (14)CO(2) and the incorporation of radioactivity into phospholipid, cholesteryl ester and diacylglycerol was unaffected by treatment with the steroid. The net output of glucose by livers from oestrogen-treated rats was impaired despite the apparent increased quantities of glycogen in the liver. The overall effect of pretreatment with oestrogen on hepatic metabolism of fatty acids is the channeling of [1-(14)C]oleate into synthesis and increased output of triacylglycerol as a moiety of the very-low-density lipoprotein, whereas ketogenesis is decreased. The effect of ethynyloestradiol on the liver is apparently independent of the nutritional state of the animal from which the liver was obtained. It is pertinent that hepatocytes prepared from livers of fed rats that had been treated with ethynyloestradiol produced fewer ketone bodies and secreted more triacylglycerol than did hepatocytes prepared from control animals. In these respects, the effects of the steroid were similar in livers from fed or starved (12-14h) rats. Oestrogens may possibly inhibit hepatic oxidation of fatty acid, making more fatty acid available for the synthesis of triacylglycerol, or may stimulate the biosynthesis of triacylglycerol, or may be active on both metabolic pathways.

Animals↗
PubMedDate not supplied

Effect of platelet-activating factor on glycogen metabolism in fetal rat lung.

Platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, or PAF) has previously been shown to induce glycogenolysis in the perfused adult rat liver and in the lung and liver of 24 day (gestational age) fetal rabbits in utero. In the present report, the effect of PAF was examined in fetal rats that were intravenously injected (through the vitellin vein) at a stage in their gestational development characterized by rapid glycogen depletion and surfactant accumulation. At 24 h after PAF administration of 2.5 micrograms and 5.0 micrograms to 19.5- and 20.5-day-old fetal rats, respectively, the lung glycogen content decreased significantly. In contrast, the inactive enantiomer of PAF did not modify the glycogenolytic response in vivo. When [14C]glucose (5 muCi) and PAF (5 micrograms) were simultaneously injected through the vitellin vein of the fetus, the radioactivity incorporated into lung glycogen was reduced as compared with control fetuses receiving the vehicle alone. An additional effect of PAF was noted in experiments designed to correlate glycogen breakdown to surfactant phospholipid biosynthesis. An inhibition of [3H]choline uptake and incorporation into phospholipids of fetal human lung explants and fetal lung type II pneumonocytes was induced by PAF. It is concluded that PAF appears to be a potential inducer of glycogen breakdown in the fetal lung and the relationship of these findings to fetal lung maturation is discussed.

Animals↗
PubMed1975

Pectin synthesis during the wall regeneration of plasmolysed tobacco leaf cells.

1. Discs of tobacco leaf lamina were floated on media containing D-[U-14C]glucose. Glucose uptake was mainly through the cut edges, and diffusion through the veins and intracellular spaces was slow. 2. Radioactivity was detected in all polysaccharide fractions extracted from the discs, including those associated with the wall. 3. Plasmolysis in sorbitol or KCL decreased the incorporation of radioactive material into all fractions. 4. Incorporation of arabinose into pectins was increased or unaffected by plasmolysis, but the incorporations of other sugars were decreased. Removal of the lower epidermis did not affect this result. 5. Seperate mechanisms for arabinan and polygalacturonorhamman syntheses must exist, and these must differ in their responses to the physiochemical, structural and organizational changes that accompany plasmolysis.

Arabinose↗
PubMed1991

Glucose entry rate in dairy cattle as determined by stable isotope 13C-labelled glucose at different stages of reproduction.

1. Uniformly labelled stable 13C-glucose was used to study glucose entry in high yielding Holstein cows (n = 8) under normal production conditions. 2. The single injection technique was repeated at three different reproductive phases. A two compartment model was applied to calculate mean entry rates of glucose resulting in: (1) Terminal phase of pregnancy (2 weeks a.p.): 0.41 g/hr/kg0.75; (2) Peak lactation (6 weeks p.p.): 0.97 g/hr/kg0.75; (3) End of lactation (37 weeks p.p.): 0.61 g/hr/kg0.75. 3. Data from studies using radioactively labelled tracers are in good agreement with our results obtained without any restrictions implied by the handling with radioactive substances.

Animals↗