The effect of serotonin on in vitro insulin secretion and biosynthesis in mice

Summary The effect of serotonin on insulin secretion and biosynthesis was studied using isolated islets of mice. Serotonin produced a small stimulatory effect on insulin secretion when glucose was present in the incubation medium at a low concentration. On the other hand, an inhibition of insulin secretion was obtained with serotonin when glucose in the medium reached 3.0 mg/ml concentration. No significant effect of serotonin was obtained on insulin biosynthesis, neither in the presence of low nor with a high glucose concentration. These results suggest that the effect of this monoamine on insulin secretion is not mediated via its effect on insulin biosynthesis.Supported by Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg, SFB 87 Ulm     

Effects of free fatty acids on insulin secretion in obesity

The prevalence of obesity in Western society has reached epidemic proportions and its aetiological role in the development of type 2 diabetes has made finding an effective treatment for the condition of crucial importance. Of the many consequences of obesity, derangements in glucose metabolism present one of the greatest problems to health. While the role of obesity in causing insulin resistance has received much attention, the effect of obesity on β‐cell failure and the consequent development of type 2 diabetes requires re‐emphasis. In this review, the current understanding of the effects of elevated free‐fatty acids on β‐cell function will be examined, including a discussion of potential mechanisms. In particular, dysregulation of biochemical pathways and alterations in key enzymes, proteins and hormones will be considered as grounds for the progression to a diabetic phenotype.     

Role of zinc in insulin biosynthesis

Summary The behaviour of proinsulin and insulin in the presence of zinc suggests it plays an important role in insulin's production in the B-cell for the vast majority of animal species. The postulate that proinsulin forms a zinc containing hexamer soon after its synthesis and that this organization of the molecule is maintained through all the subsequent processes is supported by our observation that the proinsulin hexamer is converted readily into the insulin hexamer. In addition the zinc ions enhance proinsulin's solubility and render insulin insoluble. Zinc ions also appear to play an important role in the microcrystalline character of the precipitated insulin granule. There may be advantages in condensing the stored material in this way; it will reduce contact with the surrounding membrane where the converting, and possibly other enzymes, are thought to be located, and it will tend to exclude incompletely converted hexamers.     

Effects of amino acids on pancreatic polypeptide before and after vagotomy in the dog

Summary We have previously indicated a marked influence of the vagus nerve on postprandial pancreatic polypeptide secretion. The present study was designed to determine whether the vagus nerve also plays a role in the regulation of pancreatic polypeptide secretion by absorbed nutrients. The pancreatic polypeptide responses to 17 intravenously administered amino acids, as well as arginine and glucose, were measured and compared with those 1 year after truncal vagotomy in conscious dogs. In response to the infusion of a mixture of amino acids (20 g during 60 min), plasma pancreatic polypeptide concentrations decreased in normal dogs. The effect was, however, completely reversed by vagotomy, with a significant pancreatic polypeptide release being observed (p < 0.05). Arginine (5 g during 60 min) also showed a similar, although not statistically significant, effect. After intravenous bolus-injection of glucose (0.5 g/kg body weight), a transient decrease of pancreatic polypeptide secretion was found; vagotomy abolished this response. These results suggest that the vagus nerve may have a suppressive role in the process of pancreatic polypeptide secretion induced by intravenous amino acid(s) and glucose.     

Influence of amino acids upon insulin release evoked by 3-phenylpyruvate

Summary Insulin release induced by 3-phenylpyruvate in rat pancreatic islets was unaffected by L-norleucine, L-valine, L-alanine and L-asparagine, but potentiated by L-glutamine, glycine and L-serine. The latter two amino acids also enhanced insulin secretion evoked by the combination of 3-phenylpyruvate and L-glutamine. Since 3-phenylpyruvate is known to stimulate insulin release by acting as a transamination partner and facilitating the catabolism of endogenous amino acids, the present findings suggest that the secretory efficiency of such a biochemical sequence depends upon the subcellular location of the transamination reaction and upon the interference of 3-phenylpyruvate with the transport of 2-keto acids into mitochondria.     

Effect of free fatty acids and amino acids on glucagon and insulin secretions in normal and diabetic ducks

Summary The relationship between two metabolites, free fatty acids (FFA) and amino acids (AA), and the two main pancreatic hormones, insulin and glucagon, was studied by infusing small amounts of these metabolites into normal and diabetic Peking ducks, i. e. two days after subtotal pancreatectomy. Infusion of oleic acid (0.365 g/kg/30 min as an emulsion in plasma) indicated a suppressive effect of free fatty acids on glucagon secretion, but was without effect on insulin secretion, in normal as well as in diabetic ducks, indicating that insulin might not be directly involved in the FFA-glucagon feedback in the duck. Infusions of arginine for one hour (1 g/kg/h) into normal ducks, hyperglycaemic normal birds (as a result of glucose infusion: 1 g/kg/h) and diabetic ducks, suggested the persistence of an amino acid effect on glucagon secretion, and a slight reduction of the effect on insulin secretion in diabetes. This suggests that insulin may not be involved in amino acidinduced glucagon secretion in the duck.     

Exendin 4 controls insulin production in rat islet beta cells predominantly by potentiation of glucose-stimulated proinsulin biosynthesis at the translational level

Aims/hypothesis Ideally, a therapeutic insulin secretagogue should coordinately increase insulin production and insulin secretion to maintain islet beta cell secretory capacity. We compared the incretin mimetic exendin 4 and the sulfonylurea glibenclamide (known as glyburide in the USA and Canada) for their effects in upholding a balance between (pro)insulin biosynthesis and insulin secretion in pancreatic islets.Methods Isolated rat islets were incubated for 1 or 16 h over a range of glucose concentrations (2.8–16.7 mmol/l) with or without exendin 4 (10 nmol/l) or glibenclamide (1 μmol/l). Islets were then analysed for preproinsulin mRNA expression by RNase protection and quantitative real-time RT-PCR assays. Proinsulin biosynthesis was analysed by metabolic pulse-radiolabelling, immunoprecipitation and PAGE. Insulin secretion and insulin content were analysed by radioimmunoassay.Results Neither exendin 4 nor glibenclamide affected islet preproinsulin mRNA expression. However, exendin 4 significantly increased glucose-induced proinsulin biosynthesis at the translational level within 1 h, in marked contrast to glibenclamide, which inhibited proinsulin biosynthesis, especially at basal and intermediate glucose concentrations. Exendin 4 potentiated insulin secretion in a glucose-dependent manner, whereas glibenclamide stimulated insulin secretion independently of glucose. Exendin 4 better maintained rat islet insulin content compared with glibenclamide, which depleted intracellular stores of insulin in islet beta cells by 40% within 16 h.Conclusions/interpretation Exendin 4 maintains insulin stores and beta cell secretory capacity primarily by translational control of proinsulin biosynthesis in parallel to insulin secretion. Glibenclamide does not regulate insulin production in coordination with stimulated insulin secretion, and consequently depletes islet insulin stores, compromising secretory capacity. Thus, at the level of the beta cell, incretin mimetics have an advantage over sulfonylureas for treatment of type 2 diabetes.     

Neprilysin inhibition in mouse islets enhances insulin secretion in a GLP-1 receptor dependent manner

ABSTRACT

Neprilysin, a widely expressed peptidase upregulated in type 2 diabetes, is capable of cleaving and inactivating the insulinotropic glucagon-like peptide-1 (GLP-1). Like dipeptidyl peptidase-4 (DPP-4), inhibition of neprilysin activity under diabetic conditions is associated with increased active GLP-1 levels and improved glycemic control. While neprilysin expression has been demonstrated in islets, its local contribution to GLP-1-mediated insulin secretion remains unknown. We investigated in vitro whether islet neprilysin inhibition enhances insulin secretion in response to glucose and/or exogenous GLP-1, and whether these effects are mediated by GLP-1 receptor (GLP-1R). Further, we compared the effect of neprilysin versus DPP-4 inhibition on insulin secretion. Isolated islets from wild-type (Glp1r^+/+) and GLP-1 receptor knockout (Glp1r^?/?) mice were incubated with or without the neprilysin inhibitor thiorphan and/or the DPP-4 inhibitor sitagliptin for 2.5 hours. During the last hour, insulin secretion was assessed in response to 2.8 mmol/l or 20 mmol/l glucose alone or plus exogenous active GLP-1. In Glp1r^+/+ islets, neprilysin inhibition enhanced 2.8 mmol/l and 20 mmol/l glucose- and GLP-1-mediated insulin secretion to the same extent as DPP-4 inhibition. These effects were blunted in Glp1r^?/? islets. In conclusion, inhibition of islet neprilysin in vitro increases glucose-mediated insulin secretion in a GLP-1R-dependent manner and enhances the insulinotropic effect of exogenous active GLP-1. Thus, neprilysin inhibitors may have therapeutic potential in type 2 diabetes by preserving islet-derived and circulating active GLP-1 levels.     

Effect of insulin on amino acid transport in isolated rat hepatocytes

Summary The effects of insulin on amino acid transport were studied in freshly prepared suspensions of isolated hepatocytes from adult rats. Insulin stimulated the active transport of -aminoisobutyric acid by increasing the influx. The onset of the insulin effect was delayed by thirty to sixty min. Insulin increased the Vmax of transport by about 60% without affecting the Km. Cycloheximide and actinomycin D inhibited hormonal action by 60 to 80%. Only the A system of transport was affected by insulin. Half-maximal stimulation of transport was observed with insulin at 2 to 3nmol/l, a concentration which also occupies about 50% of insulin-specific binding sites at steady state. Insulin did not antagonize the stimulatory effect of glucagon on amino acid transport.     

The effect of biguanides on secretion and biosynthesis of insulin in isolated pancreatic islets of rats

Summary Based on the clinical observation that biguanide treatment of obese patients may alter insulin levels, the influence of metformin and phenformin on basal and glucose stimulated insulin secretion, as well as on insulin biosynthesis, was studied in isolated islets of rats. — Biguanide concentrations of 100 g/ml, or higher, significantly reduced glucose stimulated insulin secretion. Both dose dependence and a difference in the intrinsic activities of metformin and phenformin were demonstrated. Incubating the same islets for a second period without biguanides, glucose stimulated insulin secretion was still decreased. Addition of glibenclamide during this second period increased insulin secretion, but did not overcome complete inhibition achieved after incubation at very high biguanide concentrations. Glucose stimulated biosynthesis of proinsulin and insulin was decreased in the presence of biguanides and completely suppressed at very high concentrations. Inhibition of cell respiration in the islet cells effected by high biguanide doses may be the reason for the inhibition of secretion and biosynthesis of insulin. — On the other hand, an insulin release was found at the highest phenformin concentration of 10 mg/ ml and during perfusion of the isolated rat pancreas with higher biguanide doses. — Biguanide concentrations found to be effective in this study are very high compared with therapeutic levels. Moreover, biguanide actions are known to be highly dependent on species, concentration and metabolic situation. — Definite conclusions from these findings regarding clinical significance, therefore, seem unwarranted.Supported by Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

Globular adiponectin augments insulin secretion from pancreatic islet beta cells at high glucose concentrations

Adiponectin plays an important role in improving insulin resistance and preventing atherosclerosis. However it has been rarely reported that adiponectin influences insulin secretion because its receptor was identified in human islet β cells. In order to investigate the direct effect of adiponectin on pancreatic islet β cells, we performed an insulin secretion test in purified rat islets, which were incubated with adiponectin (100 ng/mL) at low (3.3 mM) and high (16.7 mM) glucose concentrations. Furthermore, cell lysates were extracted from the adiponectin-treated islets for p-AMPKα assay. RTPCR and immunohistochemical examination showed both adiponectin receptor 1 (AdipoR1) and receptor 2 (A dipoR2) were expressed in islet cells and AdipoR1 was predominantly expressed. Insulin secretion was significantly increased in the presence of adiponectin for 6 h at high glucose concentration. Meanwhile, the levels of phosphorylated AMPK increased with adiponectin treatment at high glucose concentrations. It is concluded that adiponectin augments insulin secretion from pancreatic islet β cells at high glucose concentration through AMPK activation.     

Chromium-induced inhibition of insulin secretion from isolated Islets of Langerhans

Summary CrCl₃, 0.25, 0.5, 1.0, and 1.5 mmol/l inhibited glucose-induced insulin secretion in a reversible and dose dependent manner. Cr also inhibited basal secretion of insulin in the presence of 5.5 mmol/l glucose and insulin secretion stimulated by 50 mmol/l K⁺ or 15 mmol/l L-leucine. When 2 mmol/l theophylline was employed to potentiate the stimulatory effect of 16.5 mmol/l glucose, the inhibitory effect of 1.5 mmol/l Cr was reduced and that of 0.5 mmol/l Cr virtually abolished. A similar reduction in the inhibitory effect of Cr was observed when the medium calcium concentration was increased from 2.5 to 5, 7.5 and 12.5 mmol/l. Cr did not alter the conversion of ¹⁴C-glucose to ¹⁴CO₂ or ⁴⁵Ca uptake by isolated islets. It is concluded that the inhibitory effect of Cr on insulin secretion may be mediated through interference with an intracellular function of Ca⁺⁺ in the beta cell.     

Chronic effects of different non-esterified fatty acids on pancreatic islets of rats

Aims: The aim of this study was to examine the chronic effects of different non-esterified fatty acids (NEFA) on insulin secretion by pancreatic islets of normal Wistar rats in vitro. Methods: Pancreatic islets were isolated from normal Wistar rats, and were incubated with 0.2, 0.4, or 0.8 mmol/L palmitate (C16:0), stearate (C18:0), oleate (C18:1), or linoleate (C18:2) for 24 h, then the insulin secretion and pyruvate dehydrogenase (PHD) activity were examined. Results: Neither islet insulin content nor islet DNA content differed among islets incubated with each kind of NEFA. Compared with control, linoleate significantly inhibited glucose-stimulated insulin secretion (GSIS) and PDH activity at each concentration (p<0.05), while others inhibited GSIS and PDH activity significantly only at 0.4 and 0.8 mmol/L (p<0.05). There was no significant difference in GSIS and PDH activity among islets pretreated by palmitate, stearate, and oleate at the same concentration (p>0.05). However, linoleate decreased GSIS more than others at the same concentration (p<0.05), while linoleate (0.4 or 0.8 mmol/L) inhibited PDH activity more than others at the same concentration (p<0.05). Conclusions: Elevation of palmitate, stearate, oleate or linoleate decreases the β-cell secretory response to glucose, through inhibiting PDH activity. Linoleate exerts more negative effect on GSIS than other NEFA.     

Monomethylated-adenines potentiate glucose-induced insulin production and secretion via inhibition of phosphodiesterase activity in rat pancreatic islets

Monomethyladenines have effects on DNA repair, G-protein-coupled receptor antagonism and autophagy. In islet ß-cells, 3-methyladenine (3-MA) has been implicated in DNA-repair and autophagy, but its mechanism of action is unclear. Here, the effect of monomethylated adenines was examined in rat islets. 3-MA, N6-methyladenine (N6-MA) and 9-methyladenine (9-MA), but not 1- or 7-monomethylated adenines, specifically potentiated glucose-induced insulin secretion (3-4 fold; p ≤ 0.05) and proinsulin biosynthesis (∼2-fold; p ≤ 0.05). Using 3-MA as a ‘model’ monomethyladenine, it was found that 3-MA augmented [cAMP]_i accumulation (2-3 fold; p ≤ 0.05) in islets within 5 minutes. The 3-, N6- and 9-MA also enhanced glucose-induced phosphorylation of the cAMP/protein kinase-A (PKA) substrate cAMP-response element binding protein (CREB). Treatment of islets with pertussis or cholera toxin indicated 3-MA mediated elevation of [cAMP]_i was not mediated via G-protein-coupled receptors. Also, 3-MA did not compete with 9-cyclopentyladenine (9-CPA) for adenylate cyclase inhibition, but did for the pan-inhibitor of phosphodiesterase (PDE), 3-isobutyl-1-methylxanthine (IBMX). Competitive inhibition experiments with PDE-isoform specific inhibitors suggested 3-MA to have a preference for PDE4 in islet ß-cells, but this was likely reflective of PDE4 being the most abundant PDE isoform in ß-cells. In vitro enzyme assays indicated that 3-, N6- and 9-MA were capable of inhibiting most PDE isoforms found in ß-cells. Thus, in addition to known inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3′K)/m Target of Rapamycin (mTOR) signaling, 3-MA also acts as a pan-phosphodiesterase inhibitor in pancreatic ß-cells to elevate [cAMP]_i and then potentiate glucose-induced insulin secretion and production in parallel.     

Monomethylated-adenines potentiate glucose-induced insulin production and secretion via inhibition of phosphodiesterase activity in rat pancreatic islets

Monomethyladenines have effects on DNA repair, G-protein-coupled receptor antagonism and autophagy. In islet ß-cells, 3-methyladenine (3-MA) has been implicated in DNA-repair and autophagy, but its mechanism of action is unclear. Here, the effect of monomethylated adenines was examined in rat islets. 3-MA, N6-methyladenine (N6-MA) and 9-methyladenine (9-MA), but not 1- or 7-monomethylated adenines, specifically potentiated glucose-induced insulin secretion (3-4 fold; p ≤ 0.05) and proinsulin biosynthesis (～2-fold; p ≤ 0.05). Using 3-MA as a ‘model’ monomethyladenine, it was found that 3-MA augmented [cAMP]_i accumulation (2-3 fold; p ≤ 0.05) in islets within 5 minutes. The 3-, N6- and 9-MA also enhanced glucose-induced phosphorylation of the cAMP/protein kinase-A (PKA) substrate cAMP-response element binding protein (CREB). Treatment of islets with pertussis or cholera toxin indicated 3-MA mediated elevation of [cAMP]_i was not mediated via G-protein-coupled receptors. Also, 3-MA did not compete with 9-cyclopentyladenine (9-CPA) for adenylate cyclase inhibition, but did for the pan-inhibitor of phosphodiesterase (PDE), 3-isobutyl-1-methylxanthine (IBMX). Competitive inhibition experiments with PDE-isoform specific inhibitors suggested 3-MA to have a preference for PDE4 in islet ß-cells, but this was likely reflective of PDE4 being the most abundant PDE isoform in ß-cells. In vitro enzyme assays indicated that 3-, N6- and 9-MA were capable of inhibiting most PDE isoforms found in ß-cells. Thus, in addition to known inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3′K)/m Target of Rapamycin (mTOR) signaling, 3-MA also acts as a pan-phosphodiesterase inhibitor in pancreatic ß-cells to elevate [cAMP]_i and then potentiate glucose-induced insulin secretion and production in parallel.     

Isolated mouse pancreatic islets in culture: Effects of serum and different culture media on the insulin production of the islets

Summary Various conditions for tissue culture of collagenase-isolated mouse pancreatic islets were studied in an attempt to optimize the maintenance of glucose stimulated insulin biosynthesis and release in the cultured specimens. Islets which had been cultured at a physiological glucose concentration (5.5 mmol/l) in the absence of serum had an impaired glucose-stimulated insulin biosynthesis and release as well as a reduced insulin content. Thus, insulin biosynthesis was three times higher after culture in a serum supplemented medium. Further, the insulin secretion of islets cultured in the presence of serum was markedly enhanced in acute incubations with high concentrations of glucose. This response was most pronounced in islets which had been cultured free-floating. A comparison between different culture media showed that islets cultured in RPMI 1640 had the highest insulin production. The present data suggest that the most favourable conditions for long-term storage of isolated islets in culture may be obtained when the islets are maintained as free-floating explants in a culture medium consisting of RPMI 1640 supplemented with serum.     

Free fatty acids as mediators of adaptive compensatory responses to insulin resistance in dexamethasone-treated rats

BACKGROUND: Chronic low-dose dexamethasone (DEX) treatment in rats is associated to insulin resistance with compensatory hyperinsulinaemia and reduction in food intake. We tested the hypothesis that the elevation in circulating free fatty acids (FFAs) induced by DEX is the common mediator of both insulin resistance and insulin hyperproduction. METHODS: For this purpose, an anti-lipolytic agent was administered during DEX treatment to lower lipacidaemia for several hours prior to glucose and insulin tolerance tests. Leptin expression in adipose tissue (by Northern blot) and plasma leptin levels (by radioimmunoassay) were also investigated to verify whether a rise in circulating leptin could be responsible for the anorectic effect of DEX. RESULTS: Our data show that a transient pharmacological reduction of elevated plasma FFA levels abates the post-loading hyperinsulinaemia and counteracts the insulin resistance induced by DEX, supporting the hypothesis that the chronic elevation in FFAs is the common mediator of DEX-induced changes. Despite enhanced leptin expression in white adipose tissue, DEX-treated rats show no significant increase in plasma leptin levels. This suggests that the anorectic effect of DEX should be mediated, at least partially, by other factors, possibly related to the influence of concomitantly elevated plasma FFA and insulin levels on the hypothalamic centers regulating feeding. CONCLUSIONS: Our results sustain the idea that a prolonged increase in plasma FFA levels plays an important role in the adaptive regulation of glucose and energy homeostasis, not only by potentiating insulin secretion but also by providing a signal of 'nutrient abundance' capable of restraining food intake.     

Aromatic and branched-chain amino acids in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

Concentrations of the branched-chain amino acids (BCAAs) valine, leucine, and isoleucine and the aromatic amino acids (AAAs) phenylalanine and tyrosine were measured in three areas of dissected brain tissue obtained at autopsy from nine cirrhotic patients who died in hepatic encephalopathy. The controls were an equal number of subjects free from neurological, psychiatric or hepatic diseases, matched for age and time interval from death to freezing of autopsied brain samples. Amino acids were measured using high-performance liquid chromatography with fluorimetric detection. In brain tissue of cirrhotic patients, no changes in BCAA concentrations were observed compared with controls. On the other hand, phenylalanine levels were found to be increased 141% in prefrontal cortex, 86% in frontal cortex and 26% in caudate nucleus, and tyrosine content was increased by 71% in prefrontal cortex and 28% in frontal cortex with no significant increase in caudate nucleus. Alterations in the concentration of AAAs may lead to disturbances of monoamine neurotransmitters in brain. Such changes could play a role in the pathogenesis of hepatic encephalopathy resulting from chronic liver disease in man.