In vitro effects of ethanolamine on insulin secretion

The effects of ethanolamine on insulin secretion by the perfused rat pancreas were examined. During the second phase of glucose-induced insulin secretion 5-minute perfusions of ethanolamine at final concentrations of 0.1, 1 and 10 mM inhibited insulin release in a dose-related manner. When given throughout the experiment the highest dose of ethanolamine markedly suppressed both phases of glucose-induced insulin release. The inhibitory effect of ethanolamine was blunted in the presence of phentolamine. It is concluded that ethanolamine inhibits glucose-induced insulin secretion by the perfused rat pancreas and that alpha-adrenergic receptors play a role in its actions on insulin output.     

In vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid

We discovered recently in vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid. The cytotoxicity assays showed that ferulic acid (～300 μg/mL) did not cause any significant toxicity on three cell lines, platelets, leukocytes, and erythrocytes. In vitro assays showed inhibitory effects of ferulic acid on thrombin (THR)‐ or collagen/epinephrine‐stimulated platelet activation by inhibiting platelet aggregation, and decreasing clot retraction activity. The in vitro effect of ferulic acid on THR‐stimulated platelet activation was proved by the decrease in the secretion of serotonin from the platelets. The anticoagulant effects of ferulic acid were confirmed by the prolongation of the intrinsic or/and extrinsic pathways and the delay of recalcification time in plasma coagulation. Ferulic acid had antithrombotic effect in acute thromboembolism model in vivo, and decreased the expression of α_IIbβ₃/FIB and phosphorylation of AKT in THR‐stimulated platelet activation in vivo, and their antithrombotic efficacies hold promise for therapeutic targeting in our ongoing studies.     

Syntheses of ferulic acid derivatives and their suppressive effects on cyclooxygenase-2 promoter activity

Novel ferulic acid derivatives in which feruloyl groups were attached to the hydroxyl groups of myo-inositol 1,3,5-orthoformate derivatives were synthesized. These feruloyl-myo-inositols suppressed the cyclooxygenase-2 (COX-2) promoter activity in a concentration-dependent manner. Among the examined compounds, compound 9 showed the highest activity. A treatment with 100 microM of compound 9 for 24 h resulted in a 50% decrease of COX-2 promoter activity without marked cytotoxicity. Both the molecular structure in which two ferulic acid moieties are facing each other and the molecular hydrophobicity may be essential for the suppression of COX-2 promoter activity.     

Lipase-catalysed synthesis of esters of ferulic acid with natural compounds and evaluation of their antioxidant properties

Lipases from Candida antarctica (Novozyme 435^®), Candida rugosa, Chromobacterium viscosum and Pseudomonas sp. were used to perform transesterifications of vinyl ferulate with hydroxyl-steroids and p-arbutin. The antioxidant activity of the products was evaluated using the free radical 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) free radical quenching antioxidant assays, and inhibition of the oxidation of low-density lipoprotein, LDL. Arbutin ferulate was found to possess a 19% higher antiradical activity against the ABTS free radical than its precursor ferulic acid, and it also inhibited the oxidation of LDL more efficiently (by 10%) than its precursors. All of the biocatalytically synthesised products exhibited higher antioxidant activity than Trolox, the well known commercial benchmark antioxidant, and their precursor, ferulic acid.     

Synthesis of phenylalanine and production of other related compounds from phenylpyruvic acid and phenylacetic acid by ruminal bacteria,protozoa, and their mixture in vitro

Phenylalanine (Phe) synthesis and the production of other related compounds by mixed ruminal bacteria (B), protozoa (P), and a combination of the two mixture (BP) in an in vitro system were quantitatively investigated using phenylpyruvic acid (PPY) and phenylacetic acid (PAA) as substrates. Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrated mixture twice a day. Microbial suspensions were anaerobically incubated at 39 degrees C for 12 h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analysed by HPLC. A large quantity of Phe was produced from both PPY and PAA not only in B but also in P. In B suspensions, free Phe also accumulated in the medium only when PPY was used as a substrate. The ability of B to synthesize Phe from both PPY and PAA (expressed as unit 'per microbial nitrogen') was 5.1 and 24.8% higher than P, respectively. Phe production from PPY in B and P was 43.5 and 55.2% higher than that from PAA. Large amounts of PAA (17-27%) were produced from PPY in all microbial suspension and production amounts were similar in B and P. Small amounts of benzoic acid (BZA) were produced from PPY and PAA in B, P, and BP, and higher BZA production was observed in P as compared to B. Phenylpropionic acid (PPR) was produced in B from both PPY and PAA, but not in P or BP. A trace amount of phenyllactic acid (PLA) was detected only from PPY in B. Higher concentrations of an unknown compound from PPY and PAA were found to be accumulated in the body protein of B and also in the medium of P, and production of the compound from both PPY and PAA was also higher in B than P.     

Inhibitory and stimulatory effects of limb ectoderm on in vitro chondrogenesis

Previous studies have indicated possible dual effects of the limb ectoderm in cartilage differentiation. On one hand, explants from early (stage 15) wing buds are dependent on contact with the limb ectoderm for cartilage differentiation (Gumpel-Pinot, J. Embryol. Exp. Morph. 59:157-173, 1980). On the other hand, limb ectoderm from stage 23/24 wing buds inhibits cartilage differentiation by cultured limb mesenchyme cells even without direct contact (Solursh et al., Dev. Biol. 86:471-482, 1981). In the present study, ectoderms from both stage 15/16 and stage 23/24 wings are cultured under the same conditions, and ectoderms from each source are shown to have two effects. Each stimulates chondrogenesis in stage 15 wing bud mesenchyme, and each inhibits chondrogenesis in older wing mesenchyme. The results suggest that the limb ectoderm has at least dual effects on cartilage differentiation, depending on the stage of the mesenchyme. One effect involves an early mesenchymal dependence on the ectoderm. This effect requires contact between the ectoderm and mesoderm (Gumpel-Pinot, J. Embryol. Exp. Morphol. 59:157-173, 1980) but also can be observed at a distance from the ectoderm. Later, the ectoderm can act without direct contact between the ectoderm and mesoderm to inhibit chondrogenesis over some distance.     

Distribution of vasoactive intestinal polypeptide, neuropeptide-Y and substance P and their effects on insulin secretion from the in vitro pancreas of normal and diabetic rats

This study examined the pattern of distribution of vasoactive intestinal polypeptide (VIP), neuropeptide-Y (NPY) and substance P (SP) in the pancreas of diabetic rat to determine whether there are changes in the number and pattern of distribution of these neuropeptides after the onset of diabetes. Moreover, the effect of VIP, NPY and SP on insulin secretion from the pancreas of normal and diabetic rats was also examined. Diabetes mellitus (DM) was induced by a single dose of streptozotocin (STZ) given intraperitoneally (i.p.) (60 mg kg body weight(-1)). Four weeks after the induction of DM, diabetic (n = 6) and normal (n = 6) rats were anesthetized with chloral hydrate and their pancreases removed and processed for immunohistochemistry and insulin secretion. The number of insulin-positive cells in the islets of Langerhans was reduced while that of VIP and NPY increased significantly after the onset of diabetes. The pattern of distribution of VIP, NPY and SP in the nerves innervating the pancreas was similar in both normal and diabetic rats. VIP-evoked large and significant (P < 0.02) increases in insulin secretion from the pancreas of normal and diabetic rats. NPY also induced a marked (P < 0.005) increase in insulin release from pancreatic tissue fragments of normal rat. Stimulation of pancreatic tissue fragments of diabetic rat with NPY resulted in a slight but not significant increase in insulin release. SP induced a large and significant (P < 0.005) increase in insulin secretion from the pancreas of normal rat but inhibited insulin secretion significantly (P < 0.03) from isolated pancreas of diabetic rat. In summary, VIP and NPY can stimulate insulin secretion from the pancreas after the onset of diabetes. The stimulatory effect of SP on insulin secretion is reversed to inhibitory in diabetic rats.     

Acute orexin effects on insulin secretion in the rat: in vivo and in vitro studies

Orexin-A and orexin-B are members of a family of newly described orexigenic hypothalamic neuropeptides. Scanty data are available suggesting the involvement of orexins in regulation of the secretion of pituitary hormones and in control of energy homeostasis. Present studies aimed to explain whether orexins affect blood insulin concentration and insulin secretion in the rat. To check this possibility, adult female rats were subcutaneously injected with different doses (1 or 2 nmol) of orexin-A or orexin-B. A bolus administration of orexin-A resulted in an increase in blood insulin (up to min 120) and glucose (60 min after injection) concentration. The higher dose of orexin-B, on the other hand, exerted effect on insulin secretion only at min 60 of experiment and neither doses changed blood glucose level. Only orexin-A stimulated insulin secretion in an in vitro perfusion system of the rat pancreas preparation, while orexin-B was less effective. The results demonstrate that orexins belong to a group of neuropeptides influencing insulin secretion and acting directly on the pancreas. Direct, at least partial, effect of orexin on insulin secretion may be connected with the regulation of metabolism by this peptide.     

Novel 8 alpha-ergolines with inhibitory and stimulatory effects on prolactin secretion in rats

Four derivatives of the ergot dopamine (DA) agonist lisuride (LIS), namely 6-n-propyl-lisuride (6-n-propyl-LIS), transdihydrolisuride (TDHL), 6-n-propyl-transdi-hydrolisuride (6-n-propyl-TDHL) and 2-bromolisuride (2-Br-LIS) were investigated in female rats with regard to their influence on hyperprolactinaemia induced by pretreatment with reserpine (2 mg/kg i.p., 24 h) at various intervals following their subcutaneous or oral administration (0.05 mg/kg). Two hours after administration, LIS, 6-n-propyl-LIS, and 6-n-propyl-TDHL caused a statistically significant inhibition of reserpine-induced hyperprolactinaemia of about the same extent. Eight hours after administration 6-n-propyl-LIS and 6-n-propyl-TDHL were as active as after 2 h in inhibiting prolactin (PRL) secretion whereas LIS was almost ineffective in this respect. TDHL caused a statistically significant inhibition of PRL secretion at 2 and 8 h after oral administration; this effect was less pronounced after s.c. administration. In contrast to the aforementioned derivatives 2-Br-LIS further increased the reserpine-induced hyperprolactinaemia. In normal male rats pretreatment with 2-Br-LIS (0.025-6.25 mg/kg s.c., 2 h) dose-dependently stimulated PRL secretion. The present data support the assumption of the longlasting DA agonistic action of 6-n-propyl-LIS and 6-n-propyl-TDHL and of the antidopaminergic properties of 2-Br-LIS recently derived from behavioural studies.     

Regulatory effects of protein S-acylation on insulin secretion and insulin action

Post-translational modifications (PTMs) such as phosphorylation and ubiquitination are well-studied events with a recognized importance in all aspects of cellular function. By contrast, protein S-acylation, although a widespread PTM with important functions in most physiological systems, has received far less attention. Perturbations in S-acylation are linked to various disorders, including intellectual disability, cancer and diabetes, suggesting that this less-studied modification is likely to be of considerable biological importance. As an exemplar, in this review, we focus on the newly emerging links between S-acylation and the hormone insulin. Specifically, we examine how S-acylation regulates key components of the insulin secretion and insulin response pathways. The proteins discussed highlight the diverse array of proteins that are modified by S-acylation, including channels, transporters, receptors and trafficking proteins and also illustrate the diverse effects that S-acylation has on these proteins, from membrane binding and micro-localization to regulation of protein sorting and protein interactions.     

Free fatty acid accumulation in secretagogue-stimulated pancreatic islets and effects of arachidonate on depolarization-induced insulin secretion

Free fatty acids in isolated pancreatic islets have been quantified by gas chromatography-mass spectrometry after stimulation with insulin secretagogues. The fuel secretagogue D-glucose has been found to induce little change in islet palmitate levels but does induce the accumulation of sufficient unesterified arachidonate by mass to achieve an increment in cellular levels of 38-75 microM. Little of this free arachidonate is released into the perifusion medium, and most remains associated with the islets. Glucose-induced hydrolysis of arachidonate from islet cell phospholipids is reflected by release of the arachidonate metabolite prostaglandin E2 (PGE2) from perifused islets. Both the depolarizing insulin secretagogue tolbutamide (which is thought to act by inducing closure of beta-cell ATP-sensitive K+ channels and the influx of extracellular Ca2+ through voltage-dependent channels) and the calcium ionophore A23187 have also been found to induce free arachidonate accumulation within and PGE2 release from islets. Surprisingly, a major fraction of glucose-induced eicosanoid release was found not to require Ca2+ influx and occurred even in Ca(2+)-free medium, in the presence of the Ca(2+)-chelating agent EGTA, and in the presence of the Ca2+ channel blockers verapamil and nifedipine. Exogenous arachidonic acid was found to amplify the insulin secretory response of perifused islets to submaximally depolarizing concentrations of KCl, and the maximally effective concentration of arachidonate was 30-40 microM. These observations suggest that glucose-induced phospholipid hydrolysis and free arachidonate accumulation in pancreatic islets are not simply epiphenomena associated with Ca2+ influx and that arachidonate accumulation may play a role in the signaling process which leads to insulin secretion.     

The secretion of newly synthesized insulin in vitro

1. An immunological method for the purification of small quantities of insulin has been devised. 2. This method has been used to isolate labelled insulin secreted from pancreas slices incubated in vitro. The insulin had previously been labelled by incubation of the slices with [³H]leucine in vitro. 3. There is some release of labelled insulin when such slices are further incubated in media of low glucose content. When the glucose content of the medium is raised, little additional radioactive insulin is released in the first hour after labelling. However, there is a marked increase in specific radioactivity of insulin released from slices in response to a high concentration of glucose in the second and third hours. Release of labelled insulin is again diminished in the final phase, 4hr. from the start of the experiment. 4. These results are discussed in relation to possible mechanisms of insulin release from the β-cell.