Biomedical subjects
Publications and source records attributed to J Marchand.
In isolated perfused rat pancreases, the alpha-anomer of D-glucose is more potent than beta-D-glucose not solely in stimulating insulin release and suppressing glucagon output, but also in causing somatostatin secretion.
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In perifused tumoral islet cells (RINm5F line), which were prelabelled with either [32P]orthophosphate, 86Rb+ or 45Ca2+, the administration of D-glucose (1.4, 2.8 or 16.7 mM) increased the efflux of 32P, decreased the outflow of 86Rb, increased slightly the efflux of 45Ca from cells perifused in the presence of Ca2+, and decreased modestly the outflow of 45Ca from cells perifused in the absence of Ca2+. D-glucose also stimulated the net uptake of 45Ca2+. When Ba2+ (2 mM) was used, in the absence of Ca2+, instead of D-glucose as an insulin secretagogue, the efflux of 32P was little affected, but the outflow of 45Ca was dramatically increased. These changes are qualitatively similar to those occurring in normal islet cells. Nevertheless, the ionic response to D-glucose appeared, as a rule, less marked in tumoral than normal islet cells. Moreover, the concentration-response relationship was shifted to a lower range of hexose concentrations in the RINm5F cells.
Spleen cells of Biozzi HL mouse (selection V) immunized with bovine albumin-triiodothyronine conjugate were fused with P3-X63-Ag8.653 mouse myeloma cells. Thirteen monoclonal antibodies, selected by an enzyme-linked immunosorbent assay and a radioimmunoassay, were produced in mouse ascites fluid, purified and analyzed. All of them were IgG1 (kappa). The cross-reactivity of all these monoclonal antibodies with thyroxine (T4) was less than 0.2%. The association constant determined by Scatchard analysis ranged from 1.5 x 10(9) M-1 to 2.7 x 10(10) M-1.
The perfused pancreas of lean and obese Zucker rats was exposed, in the presence of L-leucine, to the anomers of D-glucose, which were administered on four successive occasions in the alpha/beta/alpha/beta or beta/alpha/beta/alpha sequence. In 5 lean and 6 obese rats, alpha-D-glucose was more efficient than beta-D-glucose in both stimulating insulin secretion and suppressing glucagon release. Although D-glucose evoked a greater release of insulin and, during prolonged exposure to the hexose, a less pronounced suppression of glucagon secretion in obese than lean rats, the anomeric specificity of these secretory responses was not different in the two groups of animals. In one lean rat, however, alpha-D-glucose, while efficiently stimulating insulin release, failed on two occasions to inhibit glucagon secretion. This isolated observation raises the possibility that the anomeric specificity of functional events evoked by D-glucose in the endocrine pancreas may occasionally be perturbed.
At a low concentration of D-glucose (3.3 mM), the phosphorylation rate of this hexose in rat pancreatic islet homogenates incubated at 8 degrees C is higher with the beta- than with the alpha-anomer, as expected from the anomeric specificity of hexokinase. In the presence of a high concentration of glucose 6-phosphate (3.0 mM), which inhibits hexokinase but not glucokinase, the phosphorylation rates of the two anomers are not significantly different from one another. Nevertheless, in intact islets exposed at 8 degrees C to the same low concentration of D-glucose, the alpha-anomer augments, more than the beta-anomer, the production of lactic acid and net uptake of 45Ca. At the same concentration (3.3 mM), the alpha-anomer is also more potent than the beta-anomer in enhancing insulin release from perfused pancreases stimulated at 37 degrees C by L-leucine or by the combination of Ba2+ and theophylline. It is concluded that the participation of glucokinase is not essential for the anomeric specificity of glycolysis and insulin release in rat pancreatic islets.
The ventral and the dorsal parts of the rat pancreas were perfused separately via either the superior mesenteric artery (0.6 ml/min) or the coeliac artery (1.4 ml/min). Control perfusions were performed via both arteries (2 ml/min). Expressed relative to the weight of tissue, the insulin content was comparable in the ventral and dorsal parts whereas the glucagon content was 2.5 times lower in the ventral than dorsal part. In comparison to the dorsal or total pancreas, the insulin secretory activity of the ventral pancreas was markedly decreased in response to either an elevation of the glucose concentration or the administration of carbamylcholine or arginine. The difference between the ventral and dorsal response was less marked at low glucose concentrations (3.3 or 7.0 mmol/l) and, possibly, in response to glucagon. In the case of glucagon release, a decreased response of the ventral pancreas was only observed when glucagon output was fully stimulated by the administration of arginine at a low glucose concentration. These results indicate that the B cell in the ventral pancreas responds poorly to several stimuli. There was little evidence to support the involvement of endogenous glucagon in the diminished sensitivity of the ventral B cells.
The effects of increasing concentrations of leucine (0.2, 2.0, and 15.0 mmol/liter) on glucagon secretion from the perfused rat pancreas were examined at various glucose levels (0, 3.3, or 8.3 mmol/liter) and in the absence or presence of either arginine (5.0 mmol/liter) or glutamine (10.0 mmol/liter). At a low glucose concentration (3.3 mmol/liter), leucine caused a dose-related biphasic increase in glucagon output in the absence of arginine, but only a transient increase in the presence of the latter amino acid. These positive responses were markedly reduced and, on occasion, abolished at a high glucose concentration (8.3 mmol/liter). Moreover, at a low glucose concentration (3.3 mmol/liter) and in the presence of arginine, the highest concentration of leucine (15.0 mmol/liter) provoked a sustained and reversible inhibition of glucagon release. Likewise, leucine (15.0 mmol/liter) reversibly inhibited glucagon secretion evoked by glutamine in the absence of glucose. Thus, leucine exerted a dual effect on the secretion of glucagon, the inhibitory effect of leucine prevailing at a high concentration of the branched chain amino acid and when glucagon secretion was already stimulated by arginine or glutamine. At a physiological concentration (0.2 mmol/liter), however, leucine was a positive stimulus for glucagon release, especially in the absence of another amino acid. Concomitantly, leucine was always a positive stimulus for both insulin and somatostatin secretion. The intimate mechanisms involved in the dual effect of leucine on glucagon secretion remain to be elucidated.
In pancreatic islet homogenates incubated in the presence of a high glucose concentration (40 mM), the beta-anomer of D-glucose is phosphorylated at a higher rate than the alpha-anomer, whether in the absence or presence of exogenous glucose 6-phosphate. However, in intact islets also exposed to 40 mM D-glucose, the production of 3H2O from D-[5-3H] glucose, the oxidation of D-[U-14C] glucose and the glucose-induced increment in either lactate production or 45Ca net uptake, as well as the release of insulin from isolated perfused pancreases, are not higher with beta- than alpha-D-glucose. It is concluded that the rate of glucose utilization by islet cells is not regulated solely by the activity of hexokinase and/or glucokinase.
The effect of glucose upon the release of glucagon and insulin from the perfused rat pancreas in vitro was studied by varying both the concentration of glucose (from 3.3 to 4.6, 8.5, or 11.1 mmol/l) and the time of exposure to an elevated concentration of the sugar (5, 10 or 23 min). The results suggest that the amount of insulin released during the early period of stimulation could contribute to both the speed and extent of the inhibition in glucagon release. The rate of recovery from inhibition in the A cell, however, appeared to be independent of insulin and was related, in a dose-dependent and time-dependent manner, only to the glucose stimulus. It is suggested that a direct effect of glucose upon the A cell is involved in the physiological regulation of glucagon secretion. An indirect effect of glucose, as mediated via insulin release, may contribute to the rapidity and magnitude of inhibition in A cell secretory activity.
Both ternary complexes consisting of lactate dehydrogenase-NAD+ and pyruvate or alpha ketobutyrate were compared, by means of circular dichroism spectra, with the corresponding binary complexes formed between lactate dehydrogenase and both NAD-pyruvate and NAD-alpha ketobutyrate adducts. Strong differences were observed. They disappeared when the ternary complexes were dissociated by addition of urea. This behaviour is explained by assuming the existence, in the abortive ternary complexes of a NAD+ activated transient form having a partial carbonium ion at position 4. Such a structure may account for the abortive and for the evolutive ternary complexes of the dehydrogenases as well as for the chemical synthesis mechanism of adducts.
The effects of K+ depletion and ouabain (0.1, 0.5, and 1 mM) on glucagon and insulin release from the in vitro perfused rat pancreas were studied in the absence or presence of arginine (10 mM). In the absence of arginine, K+ depletion resulted in an inhibition of glucagon release, both at a low (2.8 mM) and a higher (8.3 mM) concentration of glucose, whereas ouabain enhanced glucagon secretion. In the presence of arginine, K+ deprivation induced limited and short lived oscillations in the rate of glucagon release at both low and high glucose levels. In the presence of arginine, ouabain induced first a short lived stimulation and, thereafter, a severe and sustained inhibition of glucagon release. The restoration of the normal K+ concentration or the removal of ouabain was followed by a marked and transient inhibition of glucagon release which was most prominent in the presence of arginine. In agreement with previous investigations, K+ deprivation or ouabain facilitated insulin release evoked by glucose and/or arginine. The results suggest that a primary alteration in K+ availability and/or transport affects in a dual manner the release of glucagon, depending on environmental factors such as the type of stimulus used to activate the secretory process.
Recently we developed an enzyme immunoassay involving the use of steroid delta-isomerase (EC 5.3.3.1) as enzyme label and exclusion-affinity chromatography for rapid separation of free antigen-enzyme conjugate that bound to antibodies (J. Immunol. Methods 35: 267-284, 1980). Here we describe an automated version of this procedure, for immunoassay of progesterone and choriomammotropin (human placental lactogen) in serum with the use of a centrifugal analyzer. After incubation, suitable dilutions of sera or extract plus antiserum, conjugate, and double antibody were filtered on an estradiol affinity gel-filtration column; the enzyme activity of the filtrates was determined with the centrifugal analyzer. Results correlate well with those obtained by radioimmunoassay: r (progesterone) = 0.980, r (choriomammotropin) = 0.940. The within-run and between-run precision, specificity, sensitivity, accuracy, and speed of this system make it a useful tool for immunoassay.
Carbonyl compounds such as alpha-ketoglutarate, pyruvate, oxaloacetate, butyraldehyde, acetaldehyde or acetone react with NAD or NADP to give adducts. Binding studies of adducts to dehydrogenases are performed by means of ultraviolet differential spectroscopy, circular dichroism and spectrofluorimetry. The dehydrogenases show a high degree of binding specificity toward the adducts which contain their specific oxidized substrate and their specific coenzyme. The high selectivity of the dehydrogenases for adducts is evidenced by binding studies of NAD(P)-pyruvate and NAD(P)-alpha-ketoglutarate adducts on glutamate dehydrogenase at pH 7.6 and 8.9. Evidence is presented showing that adducts bind to the active site of the enzymes.
Structural analogues of the reduced coenzymes, NADH or NADPH, of dehydrogenases are prepared by addition of carbonyl compounds including: pyruvate, alpha ketoglutarate, oxaloacetate, butyraldehyde, acetaldehyde and acetone, to the oxidized coenzymes NAD(P). Some of the adducts obtained are specific inhibitors of the glutamate dehydrogenase. The specificity is related to the carbonyl compound used. The high selectivity of the dehydrogenases for adducts is evidenced by inhibition studies of NAD(P)-pyruvate and NAD(P)-alpha ketoglutarate adducts on both activities of glutamate dehydrogenase. The inhibitions are competitive with the reduced coenzymes and the oxidized substrates: adducts could be considered as structures closely related to the ternary complexes of the dehydrogenase.
Glutamate dehydrogenase is reversibly inhibited by the reaction of 1 mole of pyridoxal 5'-phosphate per mole of subunit of the enzyme polypeptide chain. We have shown that NAD(P) adducts as well as NMNH protect the glutamate dehydrogense against this reversible inactivation in the same way as reduced coenzymes. These data lead to the conclusion that it is the 1,4-dihydronicotinamide structure that is responsible for protecting the enzyme. NAD+ and NMN+ do not protect the enzyme, but in the presence of the oxidized substrate NAD+ became a good protecting agent whereas NMN+ remained ineffective. To explain the protection exerted by NAD+ in the presence of oxidized substrate, a transient activated form of the oxidized coenzyme with a 1,4-dihydronicotinamide structure and a positive charge on the C-4 atom is postulated.
The effect of calcium deprivation upon glucagon and insulin release was studied in the rat pancreas perfused in the presence of 2-ketoisocaproate (10 mM). Control perfusions were conducted in the presence of glucose (11.1 mM). In the presence of 2-ketoisocaproate, the decrease in the extracellular concentration of calcium provoked a dramatic, but reversible, enhancement of glucagon release. Such a secretory response was similar in pattern to that seen upon calcium omission in the presence of glucose. Bio-Gel P-30 chromatographic studies showed that only true glucagon (mol wt, 3500) was liberated in the pancreatic effluent during the period of calcium deprivation. On the other hand, the secretion of insulin induced by 2-ketoisocaproate was, like that elicited by glucose, markedly inhibited upon the decrease in extracellular calcium concentration (80% and 65% inhibition, respectively). The results are interpreted in support of the hypothesis that calcium may play an inhibitory role in the control of glucagon release. It is suggested that such a role is somehow linked to the metabolism of exogenous nutrients in the A2 cells.
Field potential recordings of acoustic and photic-evoked responses were obtained from 15 brain sites of freely behaving unanesthetized rats previously implanted stereotaxically with permanent electrodes. Several dosages of morphine (1, 5, 10, 30, and 50 mg/kg) were examined. The activities recorded from all the structures in this study, except the cochlear nucleus (CoN), were affected by morphine. Different sensitivities to morphine threshold were observed between structures, and several structures exhibited dose-related patterns (ventromedial hypothalamus (VMH), caudate nuucleus (CN), central gray (CG), hippocampus (Hipp), and lateral septum (Spt)). Several brain sites, after the initial dose of morphine, did not recruit more responses to subsequent doses of the drug, ie, exhibited all-or-none responses (pineal body (PB), medial thalamus (MTh), anterior hypothalamus (AH), mesencephalic reticular formation (MRF), and the dorsal raphe (DR)). In some structures, morphine induced increases in the response amplitudes, while in other sites decreases in response amplitudes were elicited. Biphasic responses, ie, increases in response amplitude after low doses of the drug and decreases in response amplitude after higher dosages, were also observed (VMH, CN, DR, CG, and MRF). The acoustic-evoked responses were affected by morphine more than the photic responses. The present observations indicated that 1) morphine exerts effects in many parts of the central nervous system (CNS); 2) some structures are more sensitive to morphine than others; 3) only a few structures exhibit dose-related patterns and, thus, may represent sites of direct morphine action; 4) some structures exhibit all-or-none responses; and 5) morphine depressed activity in some structures and increased activity in others, ie, morphine elicited different effects in different structures.