EFFECT OF INSULIN AND ADRENALINE ON CYCLIC AMP IN THE DIAPHRAGM OF NORMAL AND DIABETIC RATS
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ABSTRACT The effects of insulin and adrenaline on cyclic AMP (cAMP) levels in diaphragms of normal, streptozotocin-diabetic and insulin-treated diabetic rats were studied. Adrenaline caused a biphasic rise in cAMP with peak values of cAMP within the first few minutes. Diaphragms of diabetic rats showed an increased responsiveness to adrenaline. Injection of insulin to diabetic rats normalized the rise in cAMP after addition of adrenaline. There was no difference in basal levels of cAMP between diaphragms of normal, diabetic or insulin-treated diabetic rats. Insulin in vitro did not affect basal cAMP-levels or the release of cAMP from the tissue but significantly decreased adrenaline-induced peak levels of cAMP. This effect of insulin was abolished by theophylline. The results of the present study suggest that experimental diabetes is associated with changes of the adenylate cyclase and/or phosphodiesterase enzyme activities in skeletal muscle resulting in an increased responsiveness to adrenaline. Since insulin in vitro depressed the adrenaline-induced elevation of cAMP the increased responsiveness in diaphragms of diabetic rats might be attributed to the specific lack of insulin.Keywords:
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The significance of Ca++ for glucose stimulation of insulin release was studied in an in vitro system with beta-cell-rich pancreatic islets microdissected from oh/ob-mice. There was only a slight depression of cAMP in islets exposed to the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine after withdrawal of Ca++ from the incubation medium. The lack of a stimulatory effect of glucose noted in the absence of extracellular Ca++ is therefore probably accounted for by factors other than impaired adenylate cyclase activity. A rise of extracellular Ca++ above the concentration necessary for obtaining the optimal secretagogic effect of glucose resulted in inhibition of the glucose-stimulated insulin release, leaving basal secretions and islet contents of cAMP unaffected. Evidence was provided in support of the idea that H+ completes for Ca++ in glucose stimulation of insulin release. Both the rate of basal insulin release and that seen after stimulation with glucose were diminished by about 50% after introducing 0.2 mM La+++ in the incubation medium. These observations emphasize the significant role of Ca++ in the regulation of insulin secretion, suggesting that not only a decrease but also an increase of the functionally important intracellular pool(s) of Ca++ can result in a diminished response to glucose.
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The role of enzymes of the adenylate cyclasecAMP- protein phosphorylation system in the decreased insulin release observed during fasting has been investigated. Islets of Langerhans were isolated by collagenase digestion from paired 48-h fasted male Wistar rats and fed controls. Islets of both groups were perifused to measure rates and patterns of insulin release when exposed to 16.7 mM D-glucose or 10 mM D-glyceraldehyde. Similarly, islets from both groups were sonicated and assayed for adenylate cyclase, phosphodiesterase, protein kinase, cAMP-stimulated protein kinase, and phosphoprotein phosphatase activities. Basal insulin secretion under perifusion conditions with 2.8 mM D-glucose was not different in the two groups of islets (13.7 ± 1.6 pg insulin released islet-1 min-1 from fasted rats compared with 16.7 ± 1.2 for controls; P < 0.2). In response to 16.7 mM Dglucose, insulin release was diminished in islets from fasted animals by 50%, whereas D-glyceraldehyde-stimulated insulin release was not affected by fasting. In islets from fasted rats, basal adenylate cyclase activity was not significantly different from that observed in islets from fed controls (control, 15.3 ± 1.2 pmol cAMP mg-1 min-1; fasting, 17.6 ± 1.9; P < 0.2). Likewise, the low Km phosphodiesterase activity was not different in the fasted state (control, 5.7 ± 0.6 pmol cAMP mg-1 min-1; fasting, 5.3 ± 0.6; P < 0.3). High Km phosphodiesterase activity was decreased by 13% in islets from fasted animals (control, 425 ± 29 pmol cAMP mg-1 min-1; fasting, 369 ± 26; P < 0.02). Basal protein kinase activity in islets from fasted rats was similar to that of controls (control, 182 ± 23 pmol 32PO4 mg-1 min-1; fasted, 174 ± 24; P < 0.3). In the presence of 1 pM cAMP, activity in both groups increased by 180% (control, 502 ± 51 pmol 32PO4 mg-1 min-1; fasted, 484 ± 61; P < 0.4). Phosphoprotein phosphatase activity was decreased by 12.5% in the fasted state (control, 8.8 ± 0.5 pmol ;32PO4 mg-1 min-1; fasted, 7.7 ± 0.5; P < 0.01). From the results of these enzyme assays it appears that changed enzyme activity in the adenylate cyclase-cAMP-protein phosphorylation system plays little role in the decreased insulin release observed in the fasted state. The difference in the insulin secretory response between D-glucose and D-glyceraldehyde in islets from fasted animals supports the idea that the major ratelimiting step in stimulus-secretion coupling in the fasted state is before the metabolism of the trioses.
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ABSTRACT The effects of insulin and adrenaline on cyclic AMP (cAMP) levels in diaphragms of normal, streptozotocin-diabetic and insulin-treated diabetic rats were studied. Adrenaline caused a biphasic rise in cAMP with peak values of cAMP within the first few minutes. Diaphragms of diabetic rats showed an increased responsiveness to adrenaline. Injection of insulin to diabetic rats normalized the rise in cAMP after addition of adrenaline. There was no difference in basal levels of cAMP between diaphragms of normal, diabetic or insulin-treated diabetic rats. Insulin in vitro did not affect basal cAMP-levels or the release of cAMP from the tissue but significantly decreased adrenaline-induced peak levels of cAMP. This effect of insulin was abolished by theophylline. The results of the present study suggest that experimental diabetes is associated with changes of the adenylate cyclase and/or phosphodiesterase enzyme activities in skeletal muscle resulting in an increased responsiveness to adrenaline. Since insulin in vitro depressed the adrenaline-induced elevation of cAMP the increased responsiveness in diaphragms of diabetic rats might be attributed to the specific lack of insulin.
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Responses to glucagon from the adenylate cyclasecyclic adenosine monophosphate (cAMP) system in liver slices from control and streptozotocin-induced diabetic rats were compared. Tissue cAMP levels were similar in the basal state but responded poorly to glucagon (20 pg/ml-2 microgram/ml) in diabetic rats. Insulin treatment of diabetic rats in vivo led to a reversal of the glucagon stimulation towards the values in the control rats. The basal and glucagon-stimulated activities of adenylate cyclase in crude membrane fractions were similar in both groups. Plasma immunoreactive glucagon levels in diabetic rats were approximately three times higher than those in normal rats. Liver slices obtained from normal rats, which were injected with glucagon (0.2 mg, i.m.) 45 min previously, also showed an impaired responsiveness to glucagon of tissue cAMP levels, while no significant difference in adenylate cyclase activity was observed between the normal and glucagon-treated rats. These results suggest that the responsiveness of liver slices from the streptozotocin-induced diabetic rat has been modified by the preceding hyperglucagonemia. The reason for the observed differences between slices and crude membranes is not known.
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To investigate the mechanism of the cellular insulin insensitivity of diabetic rats, insulin binding, glucose transport, and glucose oxidation were studied in adipocytes from streptozotocin-diabetic rats. Increased insulin binding was found in cells from diabetic rats, and this was due to an increased number of insulin receptors rather than a change in receptor affinity. Basal and insulin-stimulated glucose oxidation was decreased in adipocytes from diabetic rats when the data are expressed in absolute terms or as percent increased above basal. Although the absolute rate of basal and insulin-stimulated glucose transport was decreased in adipocytes from diabetic rats, the percent increase above basal of insulin-stimulated glucose transport was not decreased. In conclusion, although the cellular insulin insensitivity exists in adipocytes from diabetic rats, the number of insulin receptors was increased, coupling between insulin receptors and the glucose transport system is intact in adipocytes from diabetic rats, and a defect in intracellular glucose metabolism rather than glucose transport plays a major role in the insulin insensitivity of adipocytes from diabetic rats.
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Abstract. In attempting to understand the causes of the hyperglycaemia observed in aging populations and to determine the mechanism(s) for the diminished in vitro insulin release from islets of Langerhans of older rats, the adenylate cyclase-cyclic AMP system was studied in isolated islets from 12 month old and 2½ month old (control) male rats to determine its role in this altered insulin secretion. Islets of Langerhans were isolated by collagenase digestion and then either incubated in the presence of low or high concentrations of glucose for studies of insulin release or were sonicated and assayed for determinations of activities of adenylate cyclase and phosphodiesterase. Insulin release was identical from islets of 12 month old and 2½ month old rats to 2.8 m m D-glucose, while in the presence of 16.7 m m D-glucose, insulin release was decreased by 33% ( P < 0.02) from islets of the older animals. Adenylate cyclase activity was diminished by 60% ( P < 0.005) from the 12 month old rats as compared with islets from the 2½ month old controls, while low K m phosphodiesterase activity was similar in islets from both groups of animals. From these studies it appears that the adenylate cyclase-cyclic AMP system may play a role in the altered insulin release from islets of aging rats.
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We studied the development of the insulin secretion mechanism in the pancreas of fetal (19- and 21-day-old), neonatal (3-day-old), and adult (90-day-old) rats in response to stimulation with 8.3 or 16.7 mM glucose, 30 mM K+, 5 mM theophylline (Theo) and 200 microM carbamylcholine (Cch). No effect of glucose or high K+ was observed on the pancreas from 19-day-old fetuses, whereas Theo and Cch significantly increased insulin secretion at this age (82 and 127% above basal levels, respectively). High K+ also failed to alter the insulin secretion in the pancreas from 21-day-old fetuses, whereas 8.3 mM and 16.7 mM glucose significantly stimulated insulin release by 41 and 54% above basal levels, respectively. Similar results were obtained with Theo and Cch. A marked effect of glucose on insulin secretion was observed in the pancreas of 3-day-old rats, reaching 84 and 179% above basal levels with 8.3 mM and 16.7 mM glucose, respectively. At this age, both Theo and Cch increased insulin secretion to close to two-times basal levels. In islets from adult rats, 8.3 mM and 16.7 mM glucose, Theo, and Cch increased the insulin release by 104, 193, 318 and 396% above basal levels, respectively. These data indicate that pancreatic B-cells from 19-day-old fetuses were already sensitive to stimuli that use either cAMP or IP3 and DAG as second messengers, but insensitive to stimuli such as glucose and high K+ that induce membrane depolarization. The greater effect of glucose on insulin secretion during the neonatal period indicates that this period is crucial for the maturation of the glucose-sensing mechanism in B-cells.
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The adenylate cyclase-cAMP system and related phosphorylating and dephosphorylating enzymes have been studied in islets of Langerhans from pregnant and nonpregnant Wistar rats to ascertain if changes in these enzymes might be related to the increase in insulin secretion observed late in pregnancy. Islets of Langerhans were isolated by collagenase digestion from 20-day-pregnant rats and age-matched female controls. Islets of each group were perifused to measure rates and patterns of insulin release when they were exposed to 2.8 mM, and subsequently 16.7 mM, Dglucose. The remaining islets were sonicated and assayed for adenylate cyclase, phosphodiesterase, phosphoprotein phosphatase, and protein kinase activities. Basal insulin secretion under perifusion conditions (2.8 mM glucose) was the same in both groups of islets (9.2 ± 2.4 pg insulin released-islet-1 min-1 from pregnant rats compared with 10.2 ± 1.8 for controls). In response to 16.7 mM glucose, both phases of insulin release were increased in islets from pregnant rats relative to controls. Within 3 min of the change from low to high glucose, the increase in insulin release in islets from pregnant rats was double that seen in control islets (P < 0.02). Summing the first and second phase responses to high glucose, the islets from pregnant rats had a 75% and 130% greater response, respectively, than did control islets. Basal adenylate cyclase activity was 13.5 ± 1.6 pmol cAMPmg-1min-1 for controls and 19.3 ± 2.1 in pregnancy (43% increase in activity, P< 0.05). High Km phosphodiesterase activity (control, 364 ± 18 pmol cAMP converted mg-1-min-1 compared with 399 ± 18) and low Km phosphodiesterase activity (control, 4.7 ± 0.4 vs. 4.1 ± 0.3 in pregnancy) were unchanged. Basal protein kinase activity was increased 34% in islets from pregnant animals (176 ± 12 compared with 131 ± 9 pmol 32PO4 incorporated ing-1 min-1, P< 0.01). cAMP (1 μM) increased the protein kinase activity by 200% in islet sonicates from both pregnant and control rats. Thus, there was a 31% increase in cAMP dependent protein kinase activity from pregnant rats (P < 0.02). Phosphoprotein phosphatase activity was measured, using [32P]spectrin II phosphate as substrate. No change in activity was observed (controls, 11.9 ± 0.7 pmol 32PO4 released mg-1-min-1vs. 12.3 ± 0.7 in pregnancy).
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The enzymes of the adenylate cyclase-cAMPprotein phosphorylation system have been studied in islets of Langerhans from older and young male Sprague-Dawley rats to ascertain if changes in these enzymes might be related to the decreased insulin release observed from islets of aging rats and to the hyperglycemia of aging. Islets of Langerhans were isolated by collagenase digestion from pancreases of 15-month-old male Sprague-Dawley rats and 3-month-old male controls. The islets were incubated in Krebs- Ringer bicarbonate buffer with low or high concentrations of glucose for studies of insulin release or were sonicated and assayed for adenylate cyclase, high and low Km phosphodiesterase, and basal protein kinase activities. Basal insulin secretion with 2.8 DIM D-glucose was not different in the two groups of islets (22.3 ± 3.6 pg insulin released islet-1 min-1 from 15-month-old rats compared with 22.5 ± 3.3 from controls; P > 0.9). In response to 16.7 mM D-glucose, insulin release was diminished in islets from older rats by 33% relative to islets from controls (95 ± 4 pg insulin released islet-1 min-1 from older rats compared with 141 ± 6 from islets of the controls; P < 0.005). Basal adenylate cyclase activity was decreased by 57% in islets from 15-month-old rats compared with islets from the young rats (17.1 ± 3.5 pmol cAMP mg-1 min-1 in islets from older rats compared with 39.3 ± 4.6 from islets of 3-month-old rats; P < 0.005). High Km phosphodiesterase activity (15-montholdrats, 221 ± 12 pmol cAMP mg-1 min-1; 3-month-old rats, 228 ± 26; P < 0.7) and low Km phosphodiesterase activity (15-monthold rats, 7.7 ± 1.3 pmol cAMP mg-1 min-1; 3-month-old rats, 6.6 ± 0.8; P < 0.2) were unchanged. Basal protein kinase activity in islets from older rats was similar to that in controls (124 ± 16 pmol 32PC>4 mg-1 min-1 for older rats compared with 122 ± 13 from young controls; P < 0.8). From the results of these enzyme assays, it appears that the adenylate cyclase-cAMP system may play a significant role in the altered insulin release from islets of aging rats.
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