Effect of beta-casomorphins and analogs on insulin release in dogs

Opioid-active substances have been isolated from bovine beta-casein peptone (beta-casomorphin). Since the ingestion of beta-casomorphin-containing foodstuff elicits an increase in postprandial insulin release, the present study was designed to determine the effect of iv infused beta-casomorphins on insulin release. The infusion of beta-casomorphin-7, -5, -4, and -3 did not alter basal insulin secretion. During prestimulation of insulin release with amino acids and glucose the infusion of beta-casomorphin-7, -5, -4, and -3 at a dose of 1 nmol/ kg . h augmented insulin release, whereas at a concentration of 100 nmol/kg . h no further stimulatory effect was observed except for the infusion of beta-casomorphin-4. In comparison, the infusion of morphine elicited a potentiation of insulin release at the lower dose, whereas no effect was observed at the higher infusion rate. Leucine-enkephalin had no effect at the lower dose but elicited an inhibitory effect at the higher rate. The infusion of opiate-active and inactive analogs of beta-casomorphin-4 resulted in a loss of its stimulatory effect, indicating that the full tetrapeptide structure of the molecule is important for its stimulatory activity on beta-cell secretion. All stimulatory and inhibitory effects of the opioid-active substances were blocked by the specific opiate-receptor antagonist naloxone. Additionally, the present data support the concept that beta-cell secretion is stimulated by mu-receptor activation and inhibited by delta-receptor activation. The K-receptor antagonist ethyl-ketocyclazocine did not alter insulin secretion. The fact that iv administered beta-casomorphins stimulate insulin release raises the possibility that ingested food-derived opioid-active substances modulate pancreatic endocrine function also after their absorption, provided the doses employed in the present study reflect physiological concentrations of circulating beta-casomorphins.     

Modulation of post-prandial insulin release by ingested opiate-like substances in dogs

Summary We have previously demonstrated that opiate-like substances in food protein (exorphins), contained in the peptic digest of gluten, stimulate insulin and glucagon release in dogs and that this effect is inhibited by the opiate antagonist naloxone. The present study was designed to evaluate the possible rôle of ingested opiate-like substances in the modulation of post-prandial insulin release. Similarly, the addition of synthetic -casomorphins, which are the opioid-active material of bovine casein peptone, elicit a stimulation of post-prandial insulin release during a liver extract-sucrose test meal. The addition of met-enkephalin to a liver extract-sucrose test meal also augmented the post-prandial insulin response. Both stimulatory effects were reversed by oral naloxone, as was the post-prandial increase of insulin following ingestion of bovine casein peptone (casopeptone). The post-prandial insulin response to digested and undigested liver extract was not affected by naloxone, suggesting that the foregoing effects are likely to be specific to opiate-like materials contained in foodstuff (exorphins). In view of previous findings, the present data are compatible with a role of opiate-like substances contained in ingested nutrients in the regulation of post-prandial insulin secretion.     

Autocrine regulation of insulin secretion

Impaired insulin secretion from pancreatic β-cells is a major factor in the pathogenesis of type 2 diabetes. The main regulator of insulin secretion is the plasma glucose concentration. Insulin secretion is modified by other nutrients, circulating hormones and the autonomic nervous system, as well as local paracrine and autocrine signals. Autocrine signalling involves diffusible molecules that bind to receptors on the same cell from which they have been released. The first transmitter to be implicated in the autocrine regulation of β-cell function was insulin itself. The importance of autocrine insulin signalling is underscored by the finding that mice lacking insulin receptors in β-cells are glucose intolerant. In addition to insulin, β-cells secrete a variety of additional substances, including peptides (e.g. amylin, chromogranin A and B and their cleavage products), neurotransmitters (ATP and γ-aminobutyric acid) and ions (e.g. zinc). Here we review the autocrine effects of substances secreted from β-cells, with a focus on acute effects in stimulus-secretion coupling, present some novel data and discuss the general significance of autocrine signals for the regulation of insulin secretion.     

Putative mediators of insulin action: regulation of pyruvate dehydrogenase and adenylate cyclase activities.

Recent evidence suggests that certain actions of insulin may be mediated by the selective generation of chemically undefined intracellular substances. Incubation of rat liver particulate fraction with low concentrations of insulin enhances the release into the supernatant of a substance that stimulates mitochondrial pyruvate dehydrogenase. Higher concentrations of insulin release less stimulating activity. It is possible to resolve activities that stimulate and inhibit pyruvate dehydrogenase by differential ethanol extraction of the supernatant solutions. The elaboration of both factors is dependent upon the presence of insulin in a dose-dependent manner. Moreover, fractions that contain the pyruvate dehydrogenase-inhibiting activity also inhibit adipocyte basal and hormonally stimulated adenylate cyclase. The production of this adenylate cyclase inhibitory activity is also stimulated by insulin. Cyclase inhibition is virtually abolished when the nonhydrolyzable ATP analog, 5'-adenylyl imidodiphosphate, is included in the assay. These results indicate that the bimodal effects of insulin on certain functions may be ascribed to the generation of at least two distinct chemical substances that show opposing activities, which may operate by regulating phosphorylation reactions.     

Effects of somatostatin on pancreatic isolated islets and peripheral tissues of rats]

This study was performed in order to evaluate the effects of somatostatin on insulin releasing mechanisms and on glucose uptake in peripheral tissues using isolated pancreatic islets, isolated rat diaphragms and epididymal fat pads. Insulin release by various concentrations of glucose were examined, and it was found that 100 ng/ml of somatostatin significantly inhibited insulin release at the glucose concentration of 200 mg/dl. Somatostatin also significantly inhibted insulin release by the administration of 5microgram/ml of glucagon with 200 mg/dl of glucose concentration and 20 mM of orginine with 200mg/dl of glucose concentrations. But at the glucose concentration of 50mg/dl, no significant inhibition of somatostatin on insulin release was observed even when various concentrations of glucagon or arginine were added. The influence of somatostatin on peripheral tissues was examined in vitro, and no significant change on glucose uptake compared with the control group was shown in either tissues. The results indicated that somatostatin directly inhibited insulin release from rat pancreatic islets but had no effect on glucose uptake in peripheral tissues. The inhibitory effect of somatostatin on insulin release may act through the common mechanism of both glucose and other substances in leading to insulin release.     

Effects of insulin infusion on endothelium-derived vasoactive substances

Summary An association between insulin resistance and hypertension has been reported in several studies. In apparent contradiction, insulin infusion in healthy volunteers is associated with vasodilatation. Furthermore, there is evidence that some insulin effects may differ between the sexes. We performed three-step hyperinsulinaemic-euglycaemic clamp studies in six men and six women to test the hypotheses that: 1) insulin might affect the release of vasoactive substances by the endothelium, and 2): this putative effect on vasoactive substances might differ between men and women. Six other women and six men served as control subjects, receiving 154 mmol/l NaCl (saline) infusion. Plasma levels of insulin, immunoreactive endothelin, l-arginine (precursor of nitric oxide), l-citrulline (by-product of nitric oxide synthesis) and cyclic GMP (second messenger of nitric oxide) were measured during infusion of insulin or 154 mmol/l NaCl (saline), respectively. We also assessed urinary excretion of 6-keto PGF-1α (a degradation product of prostacyclin reflecting prostacyclin production). Blood pressure was monitored in all subjects throughout the experiment. In women plasma levels of immunoreactive endothelin decreased from (mean ± SD) 2.58 ± 0.96 to 1.7 ± 0.72 pmol/l during insulin infusion (p < 0.01), while remaining constant in female control subjects (p < 0.02). No changes in levels of endothelin were observed in men during infusion of insulin or saline. In women levels of cGMP rose and levels of l-arginine decreased significantly during insulin infusion, consistent with an increase in nitric oxide production. Excretion of 6-keto PGF-1α also increased significantly in women during insulin infusion. No such effects were observed in men, or in women during infusion of saline. Blood pressure remained constant in all subjects during hyperinsulinaemia. We conclude that sex differences exist in the effects of insulin on the endothelium. Short-term hyperinsulinaemia in women is associated with a decline in levels of immunoreactive endothelin, and possibly with a rise in production of nitric oxide and prostacyclin. In contrast, levels of vasoactive substances remained constant in men during hyperinsulinaemia. Our findings may partly explain insulin's vasodilatory effects in healthy individuals. It remains to be investigated whether these effects are lost in insulin-resistant states. Our observation that there is a sex difference in insulin effects on the endothelium may help explain why the link between hyperinsulinaemia and cardiovascular disease appears to be clearer in men than in women. [Diabetologia (1996) 39: 1284–1292] Received: 30 April 1996 and in revised form: 18 July 1996     

Hormonal control of fat metabolism in the frog, Rana temporaria

Catecholamines, thyroxine, 5-HT, insulin, glucagon, and corticosterone were injected into cold-acclimated (5°C) summer frogs. Of these substances, only isoprenaline, 5-HT, thyroxine, and insulin significantly decreased the plasma free fatty acid (FFA) level. This effect rapidly reached its maximum in 30 min. The effects of 5-HT and thyroxine were over in 60 min, and that of isoprenaline in 180 min. Only the effect of insulin was unchanged 180 min after the injection. The effects of isoprenaline and thyroxine were blocked by adrenergic blocking agents, and that of 5-HT by methysergide. Acclimation of summer frogs to warmth (25°C) abolished the plasma FFA-level-decreasing effects of 5-HT, thyroxine, and insulin, and slightly also that of isoprenaline. In cold-acclimated winter frogs none of these hormones changed significantly the plasma FFA level. However, the effects of 5-HT and thyroxine reappeared after acclimation of animals to warmth.Only isoprenaline and 5-HT decreased the level ofplasma free glycerol, but this effect was seen only in warm-acclimated summer frogs. Only insulin caused a slight increase of short duration in the plasma free glycerol level. This effect, however, was observable only in cold-acclimated summer frogs. Because decrease of the plasma FFA level in several cases was not accompanied by a simultaneous change in the plasma free glycerol level, it can be concluded that the effective substances did not change the rate of triglyceride breakdown, but increased the oxidation and/or reesterification of FFA.     

Ghrelin secretion is inhibited by glucose load and insulin-induced hypoglycaemia but unaffected by glucagon and arginine in humans

OBJECTIVE: Circulating ghrelin levels are increased by fasting and decreased by feeding, glucose load, insulin and somatostatin. Whether hyperglycaemia and insulin directly inhibit ghrelin secretion still remains matter of debate. The aim of the present study was therefore to investigate further the regulatory effects of glucose and insulin on ghrelin secretion. DESIGN AND SUBJECTS: We studied the effects of glucose [oral glucose tolerance test (OGTT) 100 g orally], insulin-induced hypoglycaemia [ITT, 0.1 IU/kg insulin intravenously (i.v.)], glucagon (1 mg i.v.), arginine (0.5 mg/kg i.v.) and saline on ghrelin, GH, insulin, glucose and glucagon levels in six normal subjects. MEASUREMENTS: In all the sessions, blood samples were collected every 15 min from 0 up to + 120 min. Ghrelin, GH, insulin, glucagon and glucose levels were assayed at each time point. RESULTS: OGTT increased (P < 0.01) glucose and insulin while decreasing (P < 0.01) GH and ghrelin levels. ITT increased (P < 0.01) GH but decreased (P < 0.01) ghrelin levels. Glucagon increased (P < 0.01) glucose and insulin without modifying GH and ghrelin. Arginine increased (P < 0.01) GH, insulin, glucagon and glucose (P < 0.05) but did not affect ghrelin secretion. CONCLUSIONS: Ghrelin secretion in humans is inhibited by OGTT-induced hyperglycaemia and ITT but not by glucagon and arginine, two substances able to increase insulin and glucose levels. These findings question the assumption that glucose and insulin directly regulate ghrelin secretion. On the other hand, ghrelin secretion is not associated with the GH response to ITT or arginine, indicating that the somatotroph response to these stimuli is unlikely to be mediated by ghrelin.     

Ethanol influence on calcium uptake and insulin release by rat islets

Effect of acute ethanol treatment on simultaneous 45Ca++ uptake and insulin response to glucose was measured in isolated rat pancreatic islets. Ethanol, given ip 1 gm/Kg 1 h prior to sacrifice of the animal, decreased significantly 45Ca++ uptake and insulin response to 8.3 and 16.7 mM glucose. Addition of ethanol to the incubation media inhibited 45Ca++ uptake and insulin release in a dose-related matter. Ionophore A23187, which is known to enhance 45Ca++ efflux, decreased 45Ca++ uptake without affecting insulin release. Inhibitory effects of ethanol and ionophore A23187 were not additive when islets were exposed to both test substances simultaneously. Forskolin, an activator of the adenylate cyclase system potentiated the glucose mediated insulin response in rat islets. However, ethanol decreased the insulin response of islets exposed to glucose and forskolin. The data show that ethanol inhibits 45Ca++ uptake response to glucose and that ethanol influence on insulin release may involve a site beyond the formation of cyclic AMP in the process of excitation-secretion coupling.     

The effects of insulin and insulin-like materials in the brain on central cardiovascular regulation: with special reference to the central effects of sodium chloride.

The present study investigated the roles of insulin on central cardiovascular regulation. When rats were fed with a high-salt diet for 4 weeks, hypothalamic and pituitary contents of insulin-like immunoreactive substance (ILI) significantly decreased. The plasma concentration of ILI remained unaltered, however. Intracerebroventricular injections of insulin significantly decreased both the blood pressure and the heart rate, with corresponding decreases in renal sympathetic nervous activity in urethane-anesthetized rats. Microinjections of insulin into the ventromedial nucleus in the hypothalamus caused greater vasodepression and bradycardia compared with the intracerebroventricular injections. Intracerebroventricular infusions of insulin inhibited the pressor effects of simultaneously-infused hypertonic saline, and the digitalis-like immunoreactivity in both the plasma and the hypothalamus were significantly decreased by the insulin. These results indicate that ILI in the brain affects the central cardiovascular regulation through the suppression of both sympathetic nervous system activity and the release of digitalis-like substances. These mechanisms may operate particularly during a sodium load.     

Influence of insulin and of insulin resistance on platelet and vascular smooth muscle cell function

In this short review, we present the main results obtained in our laboratory in the last 15 years concerning the influence exerted by insulin on platelets and human vascular smooth muscle cells (VSMCs). In particular, we discuss: (i) the insulin ability to rapidly activate a constitutive nitric oxide synthase (NOS) in both cell types, with a consequent increase of the two nucleotides guanosine-3',5'-cyclic monophosphate (cGMP) and adenosine-3',5'-cyclic monophosphate (cAMP), well-known mediators of antiaggregation and vasodilation; (ii) the interplay of insulin with substances able to activate adenylate cyclase in both cell types; (iii) the impairment of the antiaggregating insulin effects in insulin-resistant subjects; (iv) the insulin-induced increase on endothelin in the VSMCs; (v) the insulin ability to slightly stimulate VSMC proliferation.     

Parallel insulin-like actions of human growth hormone and its part sequence hGH 7-13

The insulin-like effects of human growth hormone and the synthetic part sequence, N alpha-acetyl-hGH 7-13, on glycogen synthase and phosphorylase have been compared in an in vivo system using 16--18-day-old rats. Both the hormone and its part sequences had similar effects, increasing muscle glycogen synthase a activity and decreasing liver phosphorylase a activity, without affecting phosphorylase activity in muscle or synthase activity in liver. Insulin had similar effects, but also increased liver synthase a activity. The effects of all three substances could be abolished by prior treatment of the animals with anti-insulin serum, showing that the effects of growth hormone and its part sequences were insulin-dependent. Both growth hormone and the synthetic peptide increased the binding of insulin to liver plasma membrane. It is concluded that the insulin-like activity of human growth hormone is associated with a region containing residues 7 to 13 of the hormone molecule, and that this activity is insulin-dependent. It is suggested that both growth hormone and the synthetic peptide produce insulin-like activity by enhancing the binding of circulating insulin to its receptor.     

Studies on binding and mitogenic effect of insulin and insulin-like growth factor I in glomerular mesangial cells

The mesangial cells, as part of their smooth muscle cell function, are actively involved in regulating glomerular hemodynamics. Their overlying endothelium is fenestrated; therefore, these cells are directly exposed to plasma substances, including hormones such as insulin and insulin-like growth factor I (IGF-I). These peptides may contribute to the mesangial sclerosis and cellular hyperplasia that characterize diabetic glomerulopathy. We report herein the characterization of the receptors and the mitogenic effects of IGF-I and insulin on mouse glomerular mesangial cells in culture. The IGF-I receptor was characterized on intact cells. The Kd of the IGF-I receptor was 1.47 X 10(-9) M, and the estimated number of sites was 64,000 receptors/cell. The binding was time, temperature, and pH dependent, and the receptor showed down-regulation after exposure to serum. The expression of the receptor did not change on cells at different densities. The specific binding for insulin was too low to allow characterization of the insulin receptor on intact cells. However, it was possible to identify the insulin receptor in a wheat germ agglutinin-purified preparation of solubilized mesangial cells. This receptor showed the characteristic features of the insulin receptor, including pH dependence of binding and a curvilinear Scatchard plot. The mitogenic effects of insulin and IGF-I on mesangial cells were measured by the incorporation of [3H]thymidine into DNA. IGF-I was more potent than insulin. The half-maximal response to IGF-I stimulation occurred at 1.3 X 10(-10) M, and a similar increase with insulin was observed at concentrations in the range of 10(-7) M, suggesting that this insulin action was mediated through the IGF-I receptor. These data show that the mouse microvascular smooth muscle cells of the glomerulus express a cell surface receptor for IGF-I in vitro and that this peptide is a potent mitogen for these mesangial cells. It may, therefore, play a role in glomerular proliferative lesions. The insulin receptor is present in small numbers and does not mediate mitogenesis in mesangial cells.     

Impaired insulin secretion in human diabetes mellitus. Interactions between naloxone, phentolamine and lysine acetylsalicylate upon glucose induced insulin release

Non insulin-dependent diabetes mellitus (Type II) is characterized by the loss of the acute insulin response to glucose. Met-enkephalin, catecholamines and prostaglandin E (PGE) have all been reported to inhibit the acute insulin response to glucose in normal humans. To evaluate the hypothesis that an increased sensitivity to these endogenous substances may play a role in defective insulin secretion in diabetes, we evaluated the effects of three blocking drugs upon the impaired insulin response to glucose in Type II diabetic subjects, as well as glucose-induced insulin secretion in normal humans. In diabetics, acute insulin responses to glucose were significantly increased by all the agents tested (naloxone, phentolamine and lysine acetylsalicylate), but only the cyclooxygenase inhibitor significantly augmented second phase insulin secretion and glucose disappearance rates. The combined infusion of the three agents caused a striking increase of the acute insulin response to glucose (response before: 3 +/- 2 uU/ml; after: 22 +/- 6 uU/ml, p less than 0.01). This was accompanied by a ninefold augmentation of the second phase of insulin secretion which was the result of a synergistic interaction between the three drugs (response significantly higher than the sum of single effects). In normals, insulin responses to glucose were also significantly increased by the combined infusions of the drugs, but to a significantly lesser extent than that of diabetics. This different degree of insulin potentiation between normals and diabetics under the infusion of the three agents persisted even when the prestimulus glucose level of normals was matched to that of diabetics by a glucose infusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of the effect of various intermediate products and enzymes of carbohydrate metabolism on erythrocyte aggregation]

The effect of some substances on the aggregation of red blood cells induced by gamma-globulin and fibrinogen was studied in vitro. Fructose 1,6-diphosphate, glucose 6-phosphate, glyceraldehyde dehydrogenase, insulin and prostaglandin E1 inhibited aggregation of red cells. Glucose, glucose 1,6-phosphate and ACTH had different effects in various concentrations on red cell aggregation, while fructose, prednisolon and D-oxy-prostaglandin E1 alpha and E1 beta caused no effect.     

胰岛素增强内皮素—1的缩血管作用

为观察胰岛素对内皮素－１（ＥＴ－１）缩血管作用的影响，将高血压大鼠（ＳＨＲ）和正常血压大鼠（ＷＫＹ）的离体主动脉环置于高出生理浓度的胰岛素环境中。结果发现：ＥＴ－１对ＷＫＹ／ＳＨＲ离体血管环的作用特点是低浓度下明显收缩，随着浓度的增加收缩作用显著增强，血管去内皮后增强程度减弱；与ＷＫＹ血管相比，ＳＨＲ血管达到最大收缩的时间显著延长。加入胰岛素后，ＥＴ－１对ＷＫＹ和ＳＨＲ主动脉环收缩或舒张的反应特点相同，但作用强度明显加强，ＷＫＹ增加的最大收缩为２４％而ＳＨＲ为１６％（Ｐ＜０．０５或Ｐ＜０．０１）。认为：（１）高血压动脉血管对ＥＴ－１具有耐受性；（２）胰岛素对ＥＴ－１的缩血管作用具有增敏性，增敏程度不受血管内皮完整性的影响；（３）高胰岛素血症可通过对血管活性物质的增敏作用参与高血压病的发生和发展。     

Effects of hypothalamic factors on insulin and glucagon release from the islets of Langerhans.

Isolated pancreatic islets were used to determine whether substances of hypothalamic origin could directly influence the release of insulin and glucagon. Media in which various regions of the brain had been incubated were tested in the islet system, as were the synthetic peptides neurotensin and substance P, and the catecholamines, dopamine and norepinephrine. Substance(s) released from the ventromedial hypothalamic (VMH) segments in vitro inhibited insulin release and stimulated glucagon release from the islets. Incubates of ventrolateral hypothalamic (VLH) or cortex tissue failed to alter insulin or glucagon levels. The VMH medium retained these activities even after oxidation with K3Fe (CN)6, whereas the ability of the catecholamines to inhibit insulin release and stimulate glucagon release was eliminated by this treatment. Neurotensin and substance P (0.1 and 1.0 nmol/ml) inhibited insulin release while glucagon release was increased; however, radioimmunoassay indicated that these peptides were virtually absent from the VMH incubate. These results show that incubates of VMH contain substances which can inhibit insulin and stimulate glucagon release in vitro. They may influence the endocrine pancreas by way of the peripheral circulation although the possibility of their occurrence in or near the pancreas itself has not been excluded.     

Insulin-Like Activity of Concanavalin A and Wheat Germ Agglutinin—Direct Interactions with Insulin Receptors

Concanavalin A and wheat germ agglutinin are as effective as insulin in enhancing the rate of glucose transport and in inhibiting epinephrine-stimulated lipolysis in isolated adipocytes. These lectins, also like insulin, inhibit basal as well as epinephrine-stimulated adenylate cyclase activity of membranes obtained from homogenates of fat cells. Low concentrations of wheat germ agglutinin enhance the specific binding of insulin to receptors of fat cells and liver membranes. Higher concentrations of this plant lectin, as well as of concanavalin A, competitively displace the binding of insulin to receptors in these tissues. These effects are equally apparent in insulin-binding proteins solubilized from membranes, indicating that the plant lectins interact directly with insulin receptors. All of the effects observed with the plant lectins are reversed by simple sugars that bind specifically to these plant proteins. Agarose derivatives of the plant lectins effectively adsorb solubilized insulin-binding proteins, and these can be eluted with buffers containing specific simple sugars. The possible implications of these findings to certain biological properties (mitogenicity) of these lectins and to the mechanism of action of other growth-promoting substances are considered.     

C-peptide does not parallel increases of serum levels of substances immunologically cross-reactive with insulin in non-Hodgkin''s lymphoma patients

In 28 of 45 patients suffering from non-Hodgkin's lymphoma, the blood levels of substances detectable by the insulin-specific radioimmunoassay were supranormal, while the C-peptide levels were almost normal or even low. Such a ratio of these substances was also found in a diabetic non-Hodgkin's lymphoma patient. Two sera with the highest concentrations of these substances were investigated by gel filtration. In one case, the insulin cross-reactive material was eluted at the position of the insulin marker; in the other serum, its molecular mass was approximately 120,000.