Control of fetal insulin secretion

In this study, we investigated the way in which fetal insulin secretion is influenced by interrelated changes in blood glucose and sympathoadrenal activity. Experiments were conducted in late gestation sheep fetuses prepared with chronic peripheral and adrenal catheters. The fetus mounted a brisk insulin response to hyperglycemia but with only a minimal change in the glucose-to-insulin ratio, indicating a tight coupling between insulin secretion and plasma glucose. In well-oxygenated fetuses, alpha(2)-adrenergic blockade by idazoxan effected no change in fetal insulin concentration, indicating the absence of a resting sympathetic inhibitory tone for insulin secretion. With hypoxia, fetal norepinephrine (NE) and epinephrine secretion and plasma NE increased markedly; fetal insulin secretion decreased strikingly with the degree of change related to extant plasma glucose concentration. Idazoxan blocked this effect showing the hypoxic inhibition of insulin secretion to be mediated by a specific alpha(2)-adrenergic mechanism. alpha(2)-Blockade in the presence of sympathetic activation secondary to hypoxic stress also revealed the presence of a potent beta-adrenergic stimulatory effect for insulin secretion. However, based on an analysis of data at the completion of the study, this beta-stimulatory mechanism was seen to be absent in all six fetuses that had been subjected to a prior experimentally induced hypoxic stress but in only one of nine fetuses not subjected to this perturbation. We speculate that severe hypoxic stress in the fetus may, at least in the short term, have a residual effect in suppressing the beta-adrenergic stimulatory mechanism for insulin secretion.     

Intra-islet insulin suppresses glucagon release via GABA-GABAA receptor system

Excessive secretion of glucagon is a major contributor to the development of diabetic hyperglycemia. Secretion of glucagon is regulated by various nutrients, with glucose being a primary determinant of the rate of alpha cell glucagon secretion. The intra-islet action of insulin is essential to exert the effect of glucose on the alpha cells since, in the absence of insulin, glucose is not able to suppress glucagon release in vivo. However, the precise mechanism by which insulin suppresses glucagon secretion from alpha cells is unknown. In this study, we show that insulin induces activation of GABAA receptors in the alpha cells by receptor translocation via an Akt kinase-dependent pathway. This leads to membrane hyperpolarization in the alpha cells and, ultimately, suppression of glucagon secretion. We propose that defects in this pathway(s) contribute to diabetic hyperglycemia.     

Non-hormonal and hormonal control of glycogen metabolism in isolated sheep liver cells

1. Control of glycogen metabolism by various substrates and hormones was studied in ruminant liver using isolated hepatocytes from fed sheep. 2. In these cells glucose appeared uneffective to stimulate glycogen synthesis whereas fructose and propionate activated glycogen synthase owing to (i) a decrease in phosphorylase a activity and (ii) changes in the intracellular concentrations of glucose 6-phosphate and adenine nucleotides. 3. The activation of hepatic glycogenolysis by glucagon and alpha 1-adrenergic agents was associated with increased phosphorylase a and decreased glycogen synthase activities. 4. The simultaneous changes in these two enzyme activities suggest that in sheep liver, activation of phosphorylase a is not a prerequisite step for synthase inactivation. 5. In sheep hepatocytes, in the presence of propionate and after a lag period, insulin activated glycogen synthase without affecting phosphorylase a. 6. This latter result suggests that the direct activation of glycogen synthase by insulin is mediated by a glycogen synthase-specific kinase or phosphatase. Insulin also antagonized glucagon effect on glycogen synthesis by counteracting the rise of cAMP.     

GTP-dependent inhibition of insulin secretion by epinephrine in permeabilized RINm5F cells. Lack of correlation between insulin secretion and cyclic AMP levels

The mechanism by which alpha 2-adrenergic agonists inhibit exocytosis was investigated in electrically permeabilized insulin secreting RINm5F cells. In this preparation alpha 2-adrenoceptors remain coupled to adenylate cyclase, since basal- and forskolin-stimulated cyclic AMP production was lowered by epinephrine and clonidine by 30-50%. Cyclic AMP levels did not correlate with the rate of insulin secretion. Thus, at low Ca2+, forskolin enhanced cyclic AMP levels 5-fold without eliciting secretion, and Ca2+-stimulated secretion was associated with decreased cyclic AMP accumulation. Epinephrine (plus propranolol) inhibited Ca2+-induced insulin secretion in a GTP-dependent manner. The maximal inhibition (43%) occurred at 500 microM GTP. Clonidine also inhibited Ca2+-stimulated secretion. Replacement of GTP by GDP or by the nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)triphosphate as well as treatment of the cells with pertussis toxin prior to permeabilization abolished epinephrine inhibition of insulin secretion. Pertussis toxin did not affect Ca2+-stimulated secretion. Insulin release stimulated by 1,2-didecanoyl glycerol was also lowered by epinephrine suggesting an effect distal to the activation of protein kinase C (Ca2+/phospholipid-dependent enzyme). These results taken together with the ability of epinephrine to inhibit ionomycin-induced insulin secretion in intact cells suggest that alpha 2-adrenergic inhibition is distal to the generation of second messengers. A model is proposed for alpha 2-adrenoceptor coupling to two effector systems, namely the adenylate cyclase and the exocytotic site in insulin-secreting cells.     

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

Effects of intravenous injection of butyrate, valerate and their isomers on endocrine pancreatic responses in conscious sheep (Ovis aries)

1. The effects of intravenous injection of n-butyrate, iso-butyrate, n-valerate and iso-valerate on insulin and glucagon secretion was examined in conscious sheep. 2. Each sodium salt of the short chain fatty acids increased plasma insulin and glucagon concentrations in a dose-dependent manner (312-1250 mumol/kg body wt). 3. Both butyrate and valerate isomers with branched carbon chains had larger insulin releasing activity than isomers with straight carbon chains. 4. The glucagon responses to butyrate or valerate did not differ between the isomers with straight carbon chains and those with branched carbon chains. 5. Our results suggest that the receptive mechanism to short chain fatty acids, which may involve the nervous system, differs between the A cell and the B cell in sheep in vivo.     

Do free fatty acids induce insulin resistance in alpha cells?

Thirty years ago, Unger and Orci proposed the bihormonal-abnormality hypothesis, which highlighted that both deficient insulin secretion and excessive glucagon levels contributed to the hyperglycemic state in type 2 diabetes. The plasma free fatty acid (FFAs) concentrations are higher in patients with diabetes and prediabetes, suggesting that FFAs may be involved in the pathophysiology of diabetes. In type 2 diabetes, at least in the obese form, insulin does not seem to correct the exaggerated alpha cell responses. This phenomenon suggests that the inability of insulin to suppress the glucagon level could be caused by alpha cell insulin resistance. However, it has remained unclear whether alpha cell insulin resistance is caused by FFAs. Recent studies have demonstrated that long-term exposure to elevated FFA levels leads to hypersecretion of glucagon and accumulation of triglycerides (TG) in clonal alpha-TC1-6 cells, but the mechanism of FFA-induced alpha cell insulin resistance is unclear. We hypothesize that long-term exposure to FFAs reduces AMP-activated protein kinase (AMPK) activity and increases TG accumulation in alpha cells, leading to impaired insulin signaling of alpha cells and hypersecretion of glucagon. This hypothesis provides the first detailed examination of the effects of FFAs on alpha cells with glucagon hypersecretion. It potentially suggests that improving alpha cell insulin resistance as well as reversing lipotoxicity will normalize alpha cell function and may benefit glucose control. Consequently, AMPK and insulin-related pathways in alpha cells could be potential targets for controlling glucagon secretion and glucose counter-regulation.     

Effect of insulin on alpha1-adrenergic actions in hepatocytes from euthyroid and hypothyroid rats. Possible involvement of two pathways in alpha1-adrenergic actions

The effect of insulin on the alpha1-adrenergic stimulation of glycogenolysis and ureogenesis, which is very small or undetectable in hepatocytes from control animals, is marked in hepatocytes from hypothyroid rats; the metabolic actions due to alpha1-adrenergic activation, but not those due to glucagon, were nearly blocked by insulin in cells from hypothyroid rats. The alpha1-adrenergic-mediated stimulation of phosphatidylinositol labelling was not affected by insulin in cells from either control or hypothyroid rats. The data suggest that the alpha1-adrenergic action proceeds through two pathways, one of which is very sensitive to insulin and predominates in cells from hypothyroid rats.     

Insulin and glucagon secretion in rats: effects of calcitonin gene-related peptide

Immunoreactive calcitonin gene-related peptide (CGRP) has been shown to occur in intrapancreatic nerves and islet somatostatin cells in the rat. Therefore, we investigated the effects of CGRP on insulin and glucagon secretion in the rat. CGRP was infused i.v. at one of 3 dose levels (4.3, 17 or 68 pmol/min). Infusion of CGRP alone was found to elevate basal plasma levels of both insulin and glucagon. In contrast, CGRP impaired the plasma insulin responses to both glucose (7 mg/min; P less than 0.001) and arginine (8.5 mg/min; P less than 0.001), and inhibited the arginine-induced increase in plasma glucagon concentrations (P less than 0.001). Since CGRP and somatostatin are colocalized within the D-cells, we also infused CGRP and somatostatin together at equimolar dose levels (17 pmol/min), with glucose (7 mg/min). By that, the increase in plasma insulin concentrations decreased more rapidly than during infusion of either peptide alone. Since alpha 2-adrenoceptor activation is known to inhibit glucose-stimulated insulin secretion, we also infused CGRP together with the specific alpha 2-adrenoceptor antagonist yohimbine (37 nmol/min). In that way, the plasma insulin-lowering effect of CGRP was prevented. We have shown in the rat: (1) that CGRP stimulates basal insulin and glucagon secretion; (2) that CGRP inhibits stimulated insulin and glucagon secretion; (3) that CGRP and somatostatin more rapidly induce a potent inhibitory action on glucose-stimulated insulin secretion when given together; and (4) that the alpha 2-adrenoceptor antagonist, yohimbine, counteracts the inhibitory action of CGRP on glucose-stimulated insulin secretion. We suggest that CGRP is of importance for the regulation of insulin and glucagon secretion in the rat. The mechanisms behind the islet effects of CGRP can not be established by the present results, though they apparently require intact alpha 2-adrenoceptors.     

Effect of somatostatin suppression of glucagon secretion on glucose production in sheep.

The effect of somatostatin (SRIF) on glucagon and insulin secretion was examined in fed and fasted sheep. This was related to changes in glucose production. Infusion of SRIF at 80 micrograms/h caused a marked reduction in plasma glucagon concentrations. However, the insulin response to SRIF infusion was not consistent; its concentrations decreased occasionally, but often did not change. The depression of glucagon was not associated with a significant reduction in blood glucose concentrations in either fed or fasted sheep, but was associated with a reduction in glucose production by 12--15%. The inhibitory effect of insulin on glucose production was not markedly increased by glucagon deficiency. Infusion of insulin at 1.17 U/h with SRIF decreased glucose production only an additional 10%. Thus, it appears that under basal conditions pancreatic hormonal influences on hepatic glucose production were relatively small in sheep. This implies that under normal conditions in sheep, substrate supply has a much greater impact on hepatic glucogenesis than do hormones.     

Potentiation of arginine-induced glucagon secretion by adenosine

Adenosine and the synthetic adenosine agonists 2-chloroadenosine and N⁶-(L-2-phenylisopropyl)-adenosine were tested for effects on hormone secretion from the rat isolated perfused pancreas. These nucleosides, at concentrations of 5 μM, markedly potentiated both phases of arginine-induced glucagon release; the two synthetic agonists were more effective than adenosine. In the absence of arginine, each of the nucleosides induced a transient burst of glucagon. In contrast, adenosine and both synthetic agonists inhibited arginine-induced insulin secretion to varying degrees and caused only negligible insulin release when perfused without arginine. The adenosine antagonist 8-($\text{p}$-sulfophenyl)-theophylline prevented the actions of adenosine on hormone release from the pancreas. Our data suggest that adenosine potentiation of arginine-induced glucagon release may be mediated via adenosine receptors on alpha cell membranes; such a mechanism could provide an important endogenous control over glucagon secretion.     

Studies on the mechanism of inhibition of glucose-stimulated insulin secretion by noradrenaline in rat islets of Langerhans.

Noradrenaline (norepinephrine) was shown to be a potent inhibitor of glucose-induced insulin release from rat pancreatic islets, with half-maximal inhibition of the secretory response to 20 mM-glucose occurring at approx. 0.3 microM, and complete suppression of the response occurring at 4 microM-noradrenaline. Inhibition of insulin secretion by noradrenaline was antagonized by the alpha 2-adrenergic antagonist yohimbine (half maximally effective dose approximately 1 microM), but was largely unaffected by the alpha 1-adrenergic antagonist prazosin at concentrations up to 50 microM, suggesting that the response was mediated by alpha 2-adrenergic receptors. Noradrenaline significantly reduced the extent of 45Ca2+ accumulation in glucose-stimulated islets, although as much as 5 microM-noradrenaline was required for 50% inhibition of this response. The ability of noradrenaline to inhibit islet-cell 45Ca2+ uptake was totally abolished in media containing 1 mM-dibutyryl cyclic AMP, suggesting that the response may have been secondary to lowering of islet cyclic AMP. Under these conditions, however, noradrenaline was still able to inhibit insulin secretion maximally. The data suggest that the site(s) at which noradrenaline acts to mediate inhibition of insulin secretion in rat islets lies distal to both islet-cell cyclic AMP accumulation and Ca2+ uptake.     

A KATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans

Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca²⁺ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn²⁺ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K⁺ (K_ATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the K_ATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca²⁺ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the K_ATP channel, inhibition of voltage-gated Na⁺ (TTX) and N-type Ca²⁺ channels (ω-conotoxin), but not L-type Ca²⁺ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca²⁺ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell K_ATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

Noradrenergic Inhibition and α2-Adrenergic Stimulation of Melatonin Secretion in the Pigeon

Adrenergic regulatory mechanisms of melatonin synthesis and secretion were studied in the pigeon in vivo. Late-afternoon intraperitoneal injection of noradrenaline (NA; 1 mg/kg) resulted in a significant decrease in plasma melatonin levels in 3 h. The same effect was seen after phenylephrine treatment (1 mg/kg i.p.), indicating that an alpha 1-adrenergic mechanism may mediate the inhibition. Propranolol treatment had no effect on plasma melatonin levels, supporting this concept. Detomidine (1 mg/kg i.p.), an alpha 2-adrenergic agonist, increased melatonin levels. This stimulatory effect was blocked by yohimbine, an alpha 2-adrenergic antagonist. However, yohimbine alone had no effect on the plasma melatonin levels, suggesting that alpha 2-adrenergic transmission is not primarily responsible for the nocturnal stimulation of melatonin synthesis and secretion in the pigeon.     

Stereochemical requirements of alpha 2-adrenergic receptors for alpha-methyl substituted phenethylamines

The alpha 1- and alpha 2-adrenergic effects of the stereoisomers of alpha-methyldopamine were evaluated in guinea pig aorta and field-stimulated guinea pig ileum, respectively, in order to establish the stereochemical requirements of these receptors for alpha-methyl substituted phenethylamines. The alpha 1-adrenergic receptor did not distinguish between the stereoisomers of alpha-methyldopamine which is in marked contrast to the alpha 2-adrenergic receptor where a dramatic stereochemical preference for the 2S(+)-isomer was observed. In addition, 2R(-)-alpha-methyldopamine displayed no alpha-receptor subtype specificity whereas 2S(+)-alpha-methyldopamine was highly selective (23 fold) for the alpha 2-adrenergic receptor. These results indicate that the alpha 2-adrenergic receptor can recognize and accept methyl substituents at the alpha-carbon atom of phenethylamines when correctly oriented, while the alpha 1-adrenergic receptor cannot. Thus, the alpha-carbon atom is a major determinant of the alpha 2-adrenergic effects of phenethylamines, and plays an important role in determining alpha-receptor subtype specificity. It is hypothesized that the alpha 2-adrenergic receptor (but not alpha 1) has an additional recognition site which will accommodate alpha-substituted phenethylamines.     

Decreased alpha2-adrenergic receptor in the brain stem and pancreatic islets during pancreatic regeneration in weanling rats

Sympathetic stimulation inhibits insulin secretion. alpha(2)-Adrenergic receptor is known to have a regulatory role in the sympathetic function. We investigated the changes in the alpha(2)-adrenergic receptors in the brain stem and pancreatic islets using [(3)H]Yohimbine during pancreatic regeneration in weanling rats. Brain stem and pancreatic islets of experimental rats showed a significant decrease (p<0.001) in norepinephrine (NE) content at 72 h after partial pancreatectomy. The epinephrine (EPI) content showed a significant decrease (p<0.001) in pancreatic islets while it was not detected in brain stem at 72 h after partial pancreatectomy. Scatchard analysis of [(3)H]Yohimbine showed a significant decrease (p<0.05) in B(max) and K(d) at 72 h after partial pancreatectomy in the brain stem. In the pancreatic islets, Scatchard analysis of [(3)H]Yohimbine showed a significant decrease (p<0.001) in B(max) and K(d) (p<0.05) at 72 h after partial pancreatectomy. The binding parameters reversed to near sham by 7 days after pancreatectomy both in brain stem and pancreatic islets. This shows that pancreatic insulin secretion is influenced by central nervous system inputs from the brain stem. In vitro studies with yohimbine showed that the alpha(2)-adrenergic receptors are inhibitory to islet DNA synthesis and insulin secretion. Thus our results suggest that decreased alpha(2)-adrenergic receptors during pancreatic regeneration functionally regulate insulin secretion and pancreatic beta-cell proliferation in weanling rats.     

Effect of adenosine on the serum levels of glucose, insulin and glucagon in vivo

The in vivo effect of adenosine on the serum levels of glucose, insulin and glucagon in rats fasted for twenty four hours or after an oral glucose load were studied. Under fasting conditions adenosine produced an hyperglycaemia without change in the insulin or glucagon serum levels. After a glucose load adenosine induced a marked hyperglycaemia concomitant to a decrease in insulin serum levels and an increase in glucagon serum levels. Adenosine did not alter the relationship between insulin and glucagon. In vivo adenosine administration altered the secretion of hormones by the islets of Langerhans (increased the release of glucagon and decreased the secretion of insulin) but this was only clearly observable under stimulated conditions. Adenosine did not alter the regulatory mechanism(s) that modulate the relationship between insulin and glucagon.     

Effect of the alpha 2-blocker DG-5128 on insulin and somatostatin release from the isolated perfused rat pancreas

2[2-(4.5-Dihydro-1H-imidazol-2-yl)-1-phenylethyl] pyridine dihydrochloride sesquihydrate (DG-5128) is an alpha 2-specific-adrenergic antagonist. We have studied the effect of DG-5128 on insulin and somatostatin release from the isolated perfused rat pancrease. DG-5128 stimulated somatostatin and insulin release not only at a low glucose concentration but also at a high glucose concentration. These findings suggest that an alpha 2-adrenergic receptor plays an important role in the regulation of insulin and somatostatin secretion.     

The effects of prostaglandins on secretion of glucagon and insulin by the perfused rat pancreas.

The secretion of both glucagon and insulin by the isolated perfused rat pancreas was significantly stimulated by 10(-7) M PGH2. Experiments to show that the stimulated secretion was mediated by conversion of PGH2 to TXA2 or TXB2 revealed no correlation between the amount of secretion and the amount of thromboxane formed. Conversion of PGH2 with a crude platelet thromboxane synthase preparation caused a progressive loss of ability to secret insulin, whereas the capacity to stimulate release of glucagon remained at about one-half the maximal level. This relatively stable and selective secretagogue action on the alpha-cells appeared to be due to the formation of PGD2 by the platelet preparation. Direct administration of PGD2 confirmed this interpretation and showed clearly that this prostaglandin is a potent secretagogue for glucagon with little activity in stimulating the release of insulin. Our results have shown high and relatively equal stimulation of secretion by alpha- and beta-cells with exogenous PGE2, PGF2 alpha, and PGH2, little or no secretion by either cell type with TXA2, TXB2, or PGI2, and a unique selective stimulatory action of PGD2 upon the alpha-cell.