Stimulation Of Glucose Oxidation Research Articles

Effect of insulin on glucose oxidation and amino isobutyric acid transport and binding of insulin in chicken thymocytes

In chicken thymocytes isolated from 15–40 day-old chickens, after a 2 h incubation at 37°C, insulin stimulated amino isobutyric acid uptake (maximal response: 40–50% of increase at 1 μg insulin/ml and half maximal response at 60 ng/ml) by specifically stimulating the influx without altering the efflux. Insulin also stimulated glucose oxidation (maximal response: 11% of increase at 1 μg insulin/ml). Binding of 125I-labelled chicken insulin to thymocytes was rapid and higher at 15°C than at 37°C. At steady state, (90 min at 15°C), chicken, porcine and goose insulins were equipotent in inhibiting the binding of 125I-labelled chicken insulin. Maximal binding capacity was estimated at 1250 pg insulin/10 8 cells, i.e., 1250 binding sites/cell with an apparent dissociation constant of 200 ng insulin/ml at 15°C. Degradation of 125I-labelled chicken insulin in the incubation medium was negligible at 15°C but very noticeable at 37°C. Therefore, the low level of insulin binding at 15°C reflects a true scarcity of insulin receptors in chicken thymocytes as compared to rat thymocytes.

Calcium requirements in the action of cholecystokinin-pancreozymin on pancreatic acinar cell metabolism

The present study utilized ionophore A23187 to determine the role of Ca 2+ in pancreatic acinar cell metabolism. The ionophore A23187 in the presence of EGTA increased efflux of Ca 2+ from the rat pancreatic fragments. Ionophore and CCK-PZ were equally effective in the presence of extracellular Ca 2+ in stimulating 14C-labeled protein secretion. The ionophore decreased synthesis of new protein more effectively than CCK-PZ in the presence of extracellular Ca 2+. The effect of ionophore and CCK-PZ in combination was greater than either agent alone. Phospholipid labeling was not stimulated by A23187 in the presence of extracellular Ca 2+ in contrast to CCK-PZ. With CCK-PZ, the effect was dependent on the concentration of extracellular Ca 2+. Protein phosphorylation was stimulated ∼ 109% by CCK-PZ and ∼ 39% by ionophore. CCK-PZ stimulated protein phosphorylation in the 100,000 g supernatant whereas A23187 was ineffective. Ionophore A23187 inhibited glucose oxidation whereas CCK-PZ stimulated glucose oxidation. These data suggest that more than one kinase system might be involved in metabolic responses to hormonal stimulation of the pancreas viz. a phosphorylase kinase may be directly activated by Ca 2+ causing protein discharge whereas other kinase system may require binding of the hormone to receptor leading to other events besides protein discharge.

The role of H 2O 2 formation in the insulin-like and insulin antagonistic effects on fat cells of ω-aminoalkyl glycosides

A class of ω-aminoalkyl glycosides previously found to antagonize insulin's action on glucose oxidation in fat cells and to stimulate glucose oxidation in insulin's absence is now shown to mimic insulin also on the conversion of glucose to free fatty acids and to glycerol and glycerides. These glycosides also act like insulin by inhibiting hormone- and cholera toxin-stimulated lipolysis. Various lines of evidence demonstrate that most, if not all, of the insulin-like activity of these glycosides results from H 2O 2 formed from an amine oxidase-catalyzed oxidation of the aminoalkyl moiety of these compounds. A contaminant in the bovine plasma albumin (BPA) preparations used in the bioassays was found to represent a major source of the amine oxidase activity. Membrane (ghost) preparations were also found to possess amine oxidase activity capable of forming H 2O 2 from the glycosides in amounts sufficient to express insulin-like activity. Preliminary experiments with intact adipocytes suggest that this activity is located on the cell surface. The BPA-associated activity corresponds to the known Cu 2+-containing “plasma-type” amine oxidase (EC 1.4.3.6) on the basis of its substrate specificity and susceptibility to selective inhibitors. The plasma membrane activity appears to correspond to neither the plasma-type nor to the flavin-containing mitochondrial-type (EC 1.4.3.4) and remains to be identified. The observed potent antilipolytic effects of both H 2O 2 and the aminoalkyl glycosides points out that any mechanism used to explain the insulin-like action of H 2O 2 must account for this ability to inhibit lipolysis as well as to stimulate glucose utilization. That catalase inhibits the insulin-like action of the glycosides and H 2O 2, but not that of insulin indicates that insulin's action is not mediated by cell surface-produced H 2O 2. Also, since the insulin antagonistic activity of these glycosides was not inhibited by catalase, H 2O 2 formation is not responsible for this antagonism. The latter finding, added to present and previous evidence on the carbohydrate structural requirements involved in H 2O 2 production and in the insulin-like biological and binding properties of the aminoalkyl glycosides, is consistent with a role(s) for their carbohydrate moieties in both the insulin antagonistic and agonistic activities of these compounds.

The Insulin Receptor and Insulin of the Atlantic Hagfish: Extraordinary Conservation of Binding Specificity and Negative Cooperativity in the Most Primitive Vertebrate

The North Atlantic hagfish, a cyclostome that is representative of the most primitive vertebrates still alive, diverged from the other vertebrates about 500 million years ago. Hagfish insulin, which differs from porcine insulin in 18 (38%) of its amino acids, had a potency of 5-10% that of porcine insulin in stimulating glucose oxidation and deoxyglucose transport in rat adipocytes and was 5-10% as potent as porcine insulin in binding to insulin receptors on rat adipocytes and human (IM-9) lymphocytes. Like all other naturally occurring insulins, hagfish insulin accelerated the dissociation of 125I-porcine insulin from insulin receptors and the degree of the acceleration was related to its occupancy of the receptor. The insulin receptor of the hagfish erythrocyte showed the time, temperature, and pH dependence of binding and the negative cooperativity that are characteristic of all other insulin receptors. That the negative cooperativity is fully conserved in such an ancient insulin and receptor suggests that it is an important functional feature of this hormone-receptor system. The hagfish receptor showed the same absolute affinity and rank order of preference for insulins and insulin analogues (chicken > pork > proinsulin > guinea pig > desoctapeptide) found with other receptors of less primitive vertebrates, which supports the conclusion that the receptor for insulin is functionally better conserved evolutionary than the hormone. However, uniquely, hagfish insulin was more potent in binding to hagfish receptors than to mammalianreceptors; with all other species of insulins studied, the affinity of the hormone for homologous receptor was the same as for receptors of heterologous species.

Comparison of Several Insulin-Like Effects of Growth Hormone

Three different insulin-like effects of growth hormone were studied in segments of adipose tissue obtained from hypophysectomized rats. The onset of each response was preceded by a characteristic lag period: antilipolysis was seen only after a 10--15 minute exposure to growth hormone; stimulation of glucose oxidation was significant 20 minutes after exposure to growth hormone and increased leucine oxidation was seen only after 30 minutes. Each of the responses was measurable without a detectable delay when the tissues were exposed to hormone during a prior incubation period. Accelerated leucine oxidation was detected when 0.01 microgram/ml growth hormone was present in the incubation medium; the other responses required a minimum of 0.1 microgram/ml. Inhibitors of protein synthesis of concentrations which decreased the incorporation of 14C leucine into protein by 99% had no effect on either the antilipolytic action of growth hormone or the stimulatory action on glucose oxidation, but abolished the acceleration of leucine oxidation. In contrast to findings in diaphragm muscle, theophylline was without effect on any of the insulin-like actions of growth hormone in adipose tissue, even though it decreased the basal rate of glucose and leucine oxidation.

Synthesis and biological activity of seventeen analogues of human insulin.

We synthesized seventeen analogues of human insulin, applying the principle of stepwise, selective formation of the disulphide bonds. Most of these analogues only differ from human insulin in the replacement of a single amino acid in positions 2, 5, 6, 7, 8 and 11 of the A chain and 5, 7, 13 and 16 of the B-chain. The influence of these modifications on the physicochemical properties of the analogues is discussed. Eight analogues could be crystallized. All the analogues produce the same biological effects as insulin, but differ markedly in their potency. In isolated fat cells in vitro, [HisA8]insulin showed a relative potency of 2.46 in stimulating glucose oxidation (human insulin = 1), whereas [D-CysA6,A11]insulin had a potency of only 0.00027. Very low potency was observed when IleA2 or the half-cystines A6, A7, A11 or B7 were modified. Replacement of the invariant GlnA5 by alanine only reduced potency slightly. All the analogues are full agonists. The effects of the analogues on glucose oxidation and lipolysis are correlated, supporting the view that they are mediated by a common receptor on the fat-cell membrane. Hypoglycaemic potencies in the rat were similar to potencies in vitro. As expected, no correlation was demonstrable between antiserum binding--measured in the radioimmunoassay--and biological activity. Several results of this investigation are difficult to reconcile with the current view regarding the structure-activity relationship of insulin which appears to require further refinement.

Activity of some enzymes during growth and sporulation of Clostridium botulinum type E

The activities of alkaline and acid phosphatases, glucose dehydrogenase and NADH oxidase were assayed in cell-free extracts of sporogenic and asporogenic mutants of Clostridium botulinum. During growth of both mutants, the activities of alkaline and acid phosphatases were relatively constant, but during sporulation of the sporogenic mutant, the alkaline phosphatase activity rose to a maximum of 70 mumol/min x mg protein whereas the acid phosphatase decreased rapidly before it increased, indicating a possible role in sporogenesis. Glucose dehydrogenase activity was detected only in cell-free extracts of the sporogenic mutant and reached a maximum of 7 mumol/min x mg protein during the endospore maturation stage. The NADH oxidase activity was detected in both mutants. The NADH oxidase seems to stimulate glucose oxidation in both mutants during growth and the dehydrogenation processes of the butyric type of fermentation during spore formation in the sporogenic mutant. The findings suggest that increased glucose dehydrogenase activity in C. botulinum, as in Bacillus species, may serve as a spore event marker and that alkaline and acid phosphatases may play a regulatory role in anaerobic sporulation metabolism.

Role of phagocytosis in the activation of macrophages.

Macrophages were obtained by peritoneal lavage from untreated mice or from mice which had received either Brewer's thioglycollate broth or a suspension of streptococcus A cell walls intraperitoneally 4 days before. 3 h after harvesting, adherent cells from untreated mice were allowed to phagocytose zymosan, formaldehyde-treated sheep erythrocytes, or latex beads. Phagocytosis was stopped after 1 h and culture was continued for up to 10 days. Phagocytosis of zymosan or sheep erythrocytes triggered the immediate release of lysosomal glycosidases, stimulated the synthesis of cellular lactate dehydrogenase, and induced the delayed production and secretion of plasminogen activator . No such changes were observed upon phagocytosis of latex. Although all three particles used were phagocytosed, only zymosan and sheep erythrocytes stimulated glucose oxidation via the hexose monophosphate shunt. Similar findings were obtained in macrophages elicited with streptococcus A cell walls after zymosan phagocytosis. Thioglycollate-elicited macrophages, however, which were already secreting lysosomal hydrolases and plasminogen activator, could not be activated further by zymosan. The results of this study show that macrophages become activated after phagocytosis of particles that stimulate the activity of their hexose monophosphate shunt. The triggering event appears to be the burst of shunt activity itself or shunt-related biochemical reactions rather than phagocytic uptake per se or particle-dependent complement activation by the alternative pathway. Once initiated, macrophage activation proceeds independently of the intracellular fate of the ingested material .

Direct demonstration that receptor crosslinking or aggregation is important in insulin action

Exposure of adipocytes to antibodies to the insulin receptor results in a blockade of (125)I-labeled insulin binding, stimulation of glucose oxidation, and many more insulin-like effects. Allowing for differences in purity, antireceptor antibody is equipotent with insulin on a molar basis. Both the bivalent F(ab')(2) and monovalent Fab' fragments of the antireceptor antibody are fully active in inhibiting (125)I-labeled insulin binding. Bivalent F(ab')(2) also retains its insulin-like effects. In contrast, the monovalent Fab' loses almost all ability to stimulate glucose oxidation and acts as a competitive antagonist of insulin-stimulated glucose oxidation. Addition of anti-F(ab')(2) antisera, which crosslink the Fab'-receptor complexes, results in a restoration of the insulin-like activity of the antibody. Similarly, when cells are exposed to submaximal doses of insulin, addition of anti-insulin antibodies at low concentration enhances the biological activity of insulin. These data suggest that receptor occupancy by ligand is not sufficient for signal generation and that the insulin-like effects of antireceptor antibody (and perhaps insulin itself) require receptor aggregation or clustering. This aggregation, however, appears to be independent of microfilaments or microtubules because the insulin-like effects of antireceptor antibody, and in fact, of insulin itself, are unaffected by agents that are known to disrupt these structures.

Fluorescent labeling of hormone receptors in viable cells: preparation and properties of highly fluorescent derivatives of epidermal growth factor and insulin.

Highly fluorescent analogs of insulin and epidermal growth factor were prepared by the covalent attachment of these peptides to alpha-lactalbumin molecules that were highly substituted (i.e., seven to one) with rhodamine molecules. The alpha-lactalbumin was specifically linked to the lysine residue of insulin or to the alpha-amino group of epidermal growth factor. The insulin derivative retained 1.15% of its potency in stimulating glucose oxidation in fat cells but retained about 8.3% of its binding affinity toward receptors. The epidermal growth factor derivative was completely active in binding to fibroblast receptors and 40% as potent as the native hormone in stimulating DNA synthesis. These highly fluorescent derivatives were suitable for the specific visual labeling of receptor sites in viable cells and for measuring the lateral mobilities of the receptor-hormone complexes by fluorescent photobleaching recovery techniques. By these methods it was shown that the hormone-receptor complexes can move laterally in the plane of the plasma membrane with a diffusion coefficient of (3-5) X 10(-10) cm2/sec.

Reduced nicotinamide adenine dinucleotide phosphate oxidase in adipocyte plasma membrane and its activation by insulin: Possible role in the hormone's effects on adenylate cyclase and the hexose monophosphate shunt

An oxidase activity utilizing reduced nicotinamide adenine dinucleotide phosphate (NADPH) and producing H 2O 2 was observed in intact adipocytes of rat, as well as in the isolated plasma membranes of these cells. A stoichiometry of 1 mol of H 2O 2 production per mole of NADPH disappearance was found with isolated plasma membranes. Activation of this enzyme (R) was produced by pretreatment of cells with insulin, dithiothreitol, or sulfhydryl inhibitors, e.g., p-chloromercuribenzoate or tosyl- l-lysine chloromethyl ketone. All of these agents also stimulated glucose oxidation via the hexose monophosphate shunt. Activation of R was also observed with biologically active derivatives of insulin, e.g., proinsulin or desalanine insulin, but not with an inactive derivative, desoctapeptide insulin. The enzyme could not be activated by exposing the cells to membrane perturbants, e.g., hypotonic conditions or Triton X-100 (0.01–0.1%). The enzyme activity in the plasma membrane had a pH optimum at 6.0 and, from the Lineweaver-Burke plot, V was determined at 230 nmol and K m for NADPH was at 5.8 × 10 −5, m. The activity remained unaltered in the presence of sodium azide or cyanide. Preincubation of adipocytes with insulin or SH reagents or direct addition of oxidants, e.g., H 2O 2, potassium ferricyanide, or phenazine methosulfate, to the membranes also caused inhibition of adenylate cyclase (AC). This enzyme activity could be restored in these preparations by adding thiols. It is suggested that inhibition of AC in whole cells in response to insulin may be caused by oxidation of its SH groups by the H 2O 2 generated from the activated NADPH oxidase. Reversal of this inhibition may involve cellular reducing equivalents. The evidence suggests that the plasma membrane enzymes, i.e., NADPH oxidase and adenylate cyclase, are controlled, in part, by the intracellular redox potential.

Effects of autoantibodies to the insulin receptor on isolated adipocytes. Studies of insulin binding and insulin action.

Autoantibodies to the insulin receptor have been detected in the sera of several patients with the Type B syndrome of insulin resistance and acanthosis nigricans. In this study we have used three of these sera (B-1, B-2, and B-3) as probes of the insulin receptor in isolated rat adipocytes. Preincubation of adipocytes with each of the three sera resulted in an inhibition of subsequent [(125)I]insulin binding. 50% inhibition of binding occurred with serum dilutions of 1:5 to 1:7,500. As in our previous studies with other tissues, Scatchard analysis of the insulin-binding data was curvilinear consistent with negative cooperativity. Computer analysis suggested that in each case the inhibition of binding was due to a decrease in receptor affinity rather than a change in available receptor number. In addition to the effects on insulin binding, adipocytes pretreated with antireceptor sera also showed alterations in biological responses. All three sera produced some stimulation of basal glucose oxidation. With serum B-3, maximal stimulation of glucose oxidation occurred at a serum concentration that inhibited binding by only 10-15%, whereas with serum B-2 the dilution curves for inhibition of binding and stimulation of glucose oxidation were superimposable. Serum B-1 behaved as a partial agonist; that is, it inhibited binding more effectively than it stimulated glucose oxidation. Cells pretreated with this serum in a concentration which inhibited binding by 80% also showed a five-fold shift to the right in the dose response of insulin-stimulated glucose oxidation, whereas spermine-stimulated glucose oxidation was unaffected. Serum B-2, which contained the highest titer of antireceptor antibodies, also stimulated 2-deoxy-glucose transport, as well as glucose incorporation into lipid and glycogen. Both the ability of the serum to inhibit binding and stimulate glucose utilization were enriched in purified immunoglobulin fractions and retained in the F(ab')(2) fragment of the IgG. In addition, the bioactivity was blocked by antihuman IgG but not by anti-insulin antibodies. Enzymatic digestion of adipocytes with trypsin resulted in a complete loss of insulin-stimulated bioactivity of serum B-3, but had only minor effects on the glucose oxidation produced by serum B-1 or B-2.These data suggest that the antibodies present in these three sera bind to different determinants on the insulin receptor. Thus, these antibodies may be useful probes of receptor structure and function.

Detection, pathogenesis, and prevention of damage to human granulocytes caused by interaction with nylon wool fiber. Implications for filtration leukapheresis.

Granulocytes collected by reversible adhesion to nylon wool fiber (NWF) function relatively well in standard in vitro tests; however, they have an abnormally shortened survival time in the circulation. Assuming that this rapid disappearance represents clearance and that recognition by phagocytes is important for such clearance, we used an autologous in vitro cell:cell recognition assay to determine whether phagocytes can detect cellular changes induced by exposure of normal granulocytes to NWF. Human granulocytes incubated with NWF 1 h at 37 degrees C, eluted with 20% acid citrate dextrose plasma, and washed stimulated the hexose monophosphate shunt activity of normal granulocytes an average of twofold (193+/-40% of controls), indicating a recognition response. NWF-induced granulocyte recognition was not dependent on plasma factors or activated complement components but was dependent on the time that the granulocyte was on the NWF and was maximal by 60 min of exposure. After elution from NWF, granulocytes demonstrated resting glucose oxidation rates only slightly higher than normal; however, during the first 20 min of exposure to NWF, granulocytes increased their rate of (14)CO(2) production from [1-(14)C]glucose three- to five-fold. Therefore, experiments were performed to determine whether toxic oxygen metabolites produced by NWF-adherent cells might contribute to recognition. The results showed that (a) normal granulocytes exposed to NWF in the presence of scavengers of superoxide anion (superoxide dismutase) or free radicals (ascorbate, mannitol, or benzoate) and washed before assay did not stimulate glucose oxidation of indicator granulocytes; and (b) NWF granulocytes prepared from cells unable to generate high levels of toxic oxygen metabolites, i.e. cells prepared anaerobically or from a patient with chronic granulomatous disease, also failed to stimulate indicator granulocytes. Human granulocytes placed in contact with NWF show an oxidative burst and become recognizable to other phagocytes. Free radical scavengers are effective in minimizing this recognition conferred on NWF-procured granulocytes.

The Effect of Somatostatin on Glucose Uptake and Production by Rat Tissues in Vitro

Somatostatin has recently been used as a tool to investigate the roles of insulin and glucagon in the regulation of glucose metabolism in vivo. The alterations in glucose uptake and production that somatostatin causes have been attributed to the modifications it induces in pancreatic hormone levels rather than to effects of the peptide per se, although the direct effects of the latter on glucose turnover have not been clearly assessed. The aim of the present study therefore was to determine whether or not somatostatin directly affects either the rate of glucose uptake by fat and muscle or the rate of glucose production by the liver. Glucose uptake by the perfused rat hindquarter (skeletal muscle) was unaltered by somatostatin (10-1,000 ng./ml.) whether or not the process was stimulated by insulin (250 μU./ml.). Basal and insulin-(5-40 / μU./ml.)-stimulated glucose oxidation (uptake) by isolated rat adipocytes was also unaffected by somatostatin (5-500 ng./ml.). In addition, the peptide had no effect on basal or glucagon-(10−l0–10−9 M)-stimulated glucose output by isolated rat liver cells. Somatostatin also failed to modify the actions of these sub-maximally effective levels of glucagon on either the intracellular level of cAMP or the activity of cAMP-dependent protein kinase. The present studies support the hypothesis that the acute changes in the level and turnover of glucose induced by somatostatin are attributable to its effect on the endocrine system rather than to its direct effects on glucose metabolism.

Insulin-like activities and insulin-potentiating actions of a modified insulin B21-26 fragment: beta-Ala-Arg-Gly-Phe-Phe-Tyr-NH2.

A modified sequence of insulin B21-26, beta-Ala-Arg-Gly-Phe-Phe-Tyr-NH2 (DP-432), was synthesized and tested for its biological activities. The hexapeptide, ip injected to mice, stimulated [U-14C]-glucose incorporation into diaphragm glycogen and adipose tissue total lipid. Neither plasma glucose nor FFA (free fatty acid) was reduced by the injection. However, when injected with epinephrine, the peptide prevented a rise of plasma FFA but not that of plasma glucose due to epinephrine. In vitro, the peptide stimulated glucose oxidation and inhibited epinephrine- or isoproterenol-stimulated lipolysis of mouse adipose tissue. Furthermore, when added along with insulin to incubation media, the peptide exaggerated the effects of insulin on both glucose oxidation and isoproterenol-stimulated lipolysis. The hexapeptide thus seems to have an insulin-potentiating action as well as insulin like activities on peripheral tissue.

Metabolic changes associated with iso-osmotic regulation in brain cortex slices.

Abstract— The metabolism of single, first cortical rat brain slices was studied in response to incubation in media of various osmolalities. There was an inverse relationship between osmolality and the magnitude of the increase in tissue water content, and a direct relationship between osmolality and inulin space. Brain sodium varied directly with the media sodium, but hyperosmolal sucrose and glucose resulted in a drop in brain sodium. Brain potassium was constant in hyperosmolal sodium media, but it fell in hypo‐osmolal media and hyperosmolal sucrose and glucose media.Hypo‐osmolality depressed the oxidation of [14C]glucose to 14CO2 whereas hyperosmolality obtained with sodium, sucrose, or glucose stimulated glucose oxidation. Lactate production was enhanced only by sodium hyperosmolality. Hypo‐osmolality caused a 70% drop in phosphocreatine and a minor decrease in energy charge potential, analagous to the effects of hypoxia. Hyperosmolality had no effect on energy metabolism.The total amino acid nitrogen released into the media was suppressed by hypo‐osmolality but was increased by hyperosmolal incubation. Hyperosmolality also increased production of ammonia fourfold.The time course of the change in tissue osmolality and ion content following incubation in hyperosmolal sodium media showed the tissue osmolality reached the media osmolality within 5 min, but it took 30 min for the tissue sodium to reach equilibrium with the media sodium. This indicates that unidentified or‘idiogenic osmoles’are induced transiently by hyperosmolality associated with changes in amino acid and ammonia metabolism.

Inhibition of thyroid-stimulating hormone stimulation of protein kinase, glucose oxidation, and phospholipid synthesis in thyroid slices previously exposed to the hormone.

Prior exposure of thyroid slices to thyrotropin (TSH) induced refractoriness to subsequent stimulation of the cyclic AMP system by the hormone. Although the inhibition is incomplete, we examined whether the reduction in cyclic AMP was sufficient to alter other metabolic effects of TSH. Bovine or dog thyroid slices were incubated with or without 5-100 mU/ml TSH for 1-2h, washed, and then incubated without hormone for 1-2h. Half of the slices not exposed to TSH initially were then incubated with buffer and half were exposed to 5-100 mU/ml TSH. Slices initially incubated with TSH were also incubated with or without TSH in the third incubation. During the refractory period, TSH activation of protein kinase was inhibited even though the hormone still caused some increase in cyclic AMP concentrations. However, protein kinase activity was fully responsive to dibutyryl cyclic AMP when slices were incubated with it during the third incubation. Stimulation of glucose oxidation by TSH was significantly decreased in thyroid slices previously incubated with the hormone. During refractoriness, stimulation of glucose oxidation caused by prostaglandin E1 and dibutyryl cyclic AMP was also significantly diminished but that due to acetylcholine was not. Thus even though dibutyryl cyclic AMP could fully activate protein kinase activity during refractoriness, its effect on glucose oxidation was still inhibited, suggesting that the metabolic block responsible for this refractoriness was distal to activation of protein kinase. Stimulation of 32Pi incorporation into phospholipid by TSH and acetylcholine was also inhibited during refractoriness. Despite reduction of the stimulatory effect of TSH, binding of 125ITSH was not modified by prior incubation of thyroid slices with TSH. These results indicate that changes in the TSH receptor are not responsible for the development of refractoriness and other metabolic sites besides activation of adenylate cyclase appear to be involved.

Insulin action on glucose utilisation by chicken adipocytes

1. 1. The action of insulin on glucose metabolism by chicken adipocytes was studied. 2. 2. Insulin increased glucose uptake, glucose oxidation to CO 2 and glucose incorporation into triglyceride. 3. 3. The stimulation of glucose oxidation was the most sensitive process to insulin and concentrations down to 0.175nM stimulated carbon dioxide production. 4. 4. It was concluded that the effects of insulin on chicken adipose tissue were insufficient to account for its hypoglycaemic action in chickens.

Triacetylated insulin: biologic activity and resistance to degradation.

Tritiated N-hydroxysuccinimide acetate was prepared with specific activities up to 5 Ci/mmole and utilized to prepare tritiated triacetyl insulin. Binding of triacetyl insulin to liver plasma membranes was measured by its capacity to displace 125I-monoiodoinsulin. At low concentrations, less than 10 ng/ml triacetyl insulin appears to be as effective as native insulin in reducing the binding of 125I-monoiodoinsulin to plasma membranes. At concentrations of 20 ng/ml and higher, triacetyl insulin is significantly less effective than native insulin in displacing binding of 125I-monoiodoinsulin to plasma membranes. The properties of triacetyl insulin in this system are not ascribable to deacetylation and conversion of the substituted product to native insulin. Biologic activity of triacetylated insulin was studied in two other in vitro systmes. A comparison was made of the capacity of native beef insulin and its triacetyl derivative to stimulate glucose oxidation by epididymal fat pads. At all three concentrations tested (2, 6, and 18 ng/ml), triacetyl insulin exerted considerable activity, although its potency was significantly less than that of native insulin. Similar effects were observed when biologic activity was measured by induction of tyrosine-alpha-ketoglutarate transaminase in a cultured liver cell system where significant activity of triacetyl insulin was found at concentrations of 10(-9)-10(-7) M. In all systems tested, the activity of triacetylated insulin could not be accounted for by deacetylation and conversion to native insulin. In all systems studied, triacetyl insulin was more resistant to degradation than was monoiodoinsulin.

The ability of the calcium ionophore A-23187 to mimic some of the effects of adrenaline on the metabolism of rat submaxillary gland

Exogenous Ca*+ is required for carbamylcholine to stimulate glycogenolysis [l] and for noradrenaline to stimulate glucose oxidation [2] in rat submaxillary gland slices. It is also required for adrenaline to completely overcome the inhibition of glucose utilization caused by acetoacetate [3]. This latter effect of adrenaline is associated with a decrease of tissue [ATP] which occurs only when exogenous Ca*+ is present [3] . These findings suggest that Ca2+ might be involved in the mechanism by which adrenaline exerts some of its metabolic effects. Ca*+ may simply be required for the hormone to bind to its receptor in the membrane or the hormonereceptor interaction may stimulate movement of Ca*+ into the cell and thus initiate the metabolic response. The ionophore A-23 187 is capable of facilitating the movement of Ca*‘and other divalent cations across biological membranes [4] and can be used as a tool to investigate whether changes in Ca*+ flux can alter cellular metabolism. Indeed A-23187 stimulates glycogen breakdown in submaxillary gland slices [S] and the results reported here show that A-23 187 can also stimulate glucose uptake and cause the tissue [ATP] to decrease. On the other hand, adrenaline was no longer able to stimulate glucose metabolism in the presence of tetracaine or Ni*‘, which appear’ to inhibit Ca*+ flux into cells.