Novel pharmacological approaches to the treatment of type 2 diabetes.

The huge increase in type 2 diabetes is a burden worldwide. Many marketed compounds do not address relevant aspects of the disease; they may already compensate for defects in insulin secretion and insulin action, but loss of secreting cells (ß-cell destruction), hyperglucagonemia, gastric emptying, enzyme activation/inhibition in insulin-sensitive cells, substitution or antagonizing of physiological hormones and pathways, finally leading to secondary complications of diabetes, are not sufficiently addressed. In addition, side effects for established therapies such as hypoglycemias and weight gain have to be diminished. At present, nearly 1000 compounds have been described, and approximately 180 of these are going to be developed (already in clinical studies), some of them directly influencing enzyme activity, influencing pathophysiological pathways, and some using G-protein-coupled receptors. In addition, immunological approaches and antisense strategies are going to be developed. Many compounds are derived from physiological compounds (hormones) aiming at improving their kinetics and selectivity, and others are chemical compounds that were obtained by screening for a newly identified target in the physiological or pathophysiological machinery. In some areas, great progress is observed (e.g., incretin area); in others, no great progress is obvious (e.g., glucokinase activators), and other areas are not recommended for further research. For all scientific areas, conclusions with respect to their impact on diabetes are given. Potential targets for which no chemical compound has yet been identified as a ligand (agonist or antagonist) are also described.

The impact of ANG II and IV on INS-1 cells and on blood glucose and plasma insulin.

The impact of angiotensin (ANG) for peripheral, global effects is well known. Local ANG systems including that of the insulin-releasing beta cell are not well investigated. In insulin-secreting cell line (INS-1), AT(1) and AT(4) receptors for ANG II and IV were demonstrated by Western blots. Only small amounts of ANG II-binding sites of low affinity were observed. ANG II and SARILE displaced binding of (125)I-ANG II. ANG II and IV as well as their non-degradable analogs SARILE and Nle-ANG IV increased the glucose-induced insulin release in a bell-shaped way; the maximum effect was at approximately 1 nM. The increase was antagonized by 1 microM losartan or 10 microM divalinal (AT(1) and AT(4) receptor antagonists, respectively). The insulin release was accompanied by a (45)Ca(2+) uptake in the case of ANG II and ANG IV. Divalinal abolished the effect of ANG IV and Nle-ANG IV on this parameter. ANG IV reduced the increase in blood glucose during a glucose tolerance test with corresponding, albeit smaller effects on plasma insulin. Using confocal laser scanning microscopy, transfected insulin-regulated aminopeptidase (IRAP) with AT(4) receptors was shown to be accumulated close to the nucleus and the cytosolic membrane, whereas GLUT4 was not detectable. IRAP was inhibited by ANG IV. In conclusion, AT(1) and AT(4) receptors may be involved in diabetic homeostasis. Effects are mediated by insulin release, which is accompanied by an influx of extracellular Ca(2+). The impact of ANG IV/IRAP agonists may be worth being used as antidiabetics.

Novel therapeutics for type 2 diabetes: incretin hormone mimetics (glucagon-like peptide-1 receptor agonists) and dipeptidyl peptidase-4 inhibitors.

Known treatments of type 2 diabetes mellitus have limitations such as weight gain, and hypoglycaemias. A new perspective is the use of incretin hormones and incretin enhancers. Incretins are defined as being responsible for the higher insulin release after an oral glucose load compared to an intravenous glucose load. The delicate balance of glucose homeostasis, in which incretin hormones are involved, is disturbed in type 2 diabetes mellitus. The incretin GLP-1 helps to maintain glucose homeostasis through stimulation of insulin secretion and inhibition of glucagon release in a glucose-dependent manner. This is associated with reductions in body weight, and no risk of hypoglycaemias. When classical oral agents have failed to maintain adequate glycaemic control, incretin mimetics may be of particular value for obese patients and those who have little control over meal sizes. Exenatide was marketed as a GLP-1 analogue and longer acting incretin mimetics such as liraglutide, albiglutide and others have the same pharmacological profile. In addition to incretin mimetics incretin enhancers which inhibit/delay degradation of incretins were developed: so-called DPP-4 inhibitors such as sitagliptin and vildagliptin are approved in Europe. Their differences from incretin mimetics include: oral bioavailability, less side effects with overdose, no direct CNS effects (nausea and vomiting) and no effect on weight. In rodent models of diabetes, but not yet in humans, GLP-1 receptor agonists and DPP-4 inhibitors increase islet mass and preserve beta-cell function. Incretin mimetics and enhancers expand type 2 diabetes treatment, are still not first line therapy and it is discussed if they are to be prophylactically used.

Pharmacological properties of the extract and some isolated compounds of Clausena lansium stem bark: anti-trichomonal, antidiabetic, anti-inflammatory, hepatoprotective and antioxidant effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Clausena lansium (Fool's Curry Leaf) is used for various ethnomedical conditions in some countries, including bronchitis, malaria, viral hepatitis, acute and chronic gastro-intestinal inflammation, and as a spicy substitute of the popular Curry leaf tree (Murraya koenigii). AIM OF THE STUDY: This study was to evaluate the ethnomedical uses of the stem bark in inflammatory conditions, hepatotoxicity and to determine the anti-diabetic and anti-trichomonal properties of the plant. MATERIALS AND METHOD: Anti-trichomonal, in vivo and in vitro antidiabetic and insulin stimulating, anti-inflammatory, hepatoprotective and anti-oxidant activities using Trichomonas gallinae, glucose loaded rats and in vitro insulin secreting cell line (INS-1 cell), carrageenin-induced rat paw oedema, CCl(4)-induced hepatotoxicity and DPPH scavenging ability methods respectively for the extracts and some isolates were determined. RESULTS: A dichloromethane extract was superior over methanolic extract with respect to an anti-trichomonal activity which was measured after 24 and 48 h. The isolated compounds imperatorin and 3-formylcarbazole had the main anti-trichomonal activity (LC(50)s of 6.0, 3.0 and 3.6, 9.7 microg/mL after 24 and 48 h, respectively). Methanolic extract (100 mg/kg) induced maximum and significant (p<0.05) anti-hyperglycaemic activity of 15.8% at 30 min and a 38.5% increase in plasma insulin at 60 min, compared to control. The increase in plasma insulin after 60 min, compared to 0 min, was 62.0% (p<0.05). The significant 174.6% increase of insulin release from INS-1 cells (in vitro) at 0.1 mg/ml indicates that it mediates its antidiabetic action mainly by stimulating insulin release. Imperatorin and chalepin were the major active constituents increasing in vitro insulin release to 170.3 and 137.9%, respectively. 100 mg/kg of the methanolic extract produced an anti-inflammatory activity after 4 h. A sedative effect was not observed. 100 and 200 mg/kg of methanolic extract administered i.p., reduced CCl4-induced hepatotoxicity firstly by 5.3 and 8.4% reduction in phenobarbitone-sleeping time respectively, secondly by reversing the reduction in serum liver proteins by 7.0-8.8%, serum AST, ALT and ALP activities by 27.7-107.9% and thirdly by diminishing increased values of plasma AST, ALT and ALP activities by 13.2-83.8%. The extract exhibited antioxidant activities. CONCLUSION: The hepatoprotective activity of C. lansium is partly due to its anti-oxidant and anti-inflammatory properties and confirms its folkloric use in the treatment of gastro-intestinal inflammation, bronchitis and hepatitis. In addition the use of C. lansium stem bark would be useful in diabetes and trichomoniasis.

Modulation of insulin release by adenosine A1 receptor agonists and antagonists in INS-1 cells: the possible contribution of 86Rb+ efflux and 45Ca2+ uptake.

Due to the lack of specific agonists and antagonists the role of adenosine receptor subtypes with respect to their effect on the insulin secretory system is not well investigated. The A1 receptor may be linked to different 2nd messenger systems, i.e. cAMP, K+- and 45Ca2+ channel activity. Partial A1 receptor agonists are going to be developed in order to improve diabetes (increase in insulin sensitivity, lowering of FFA and triglycerides). In this study newly synthesized selective A1 receptor agonists and antagonists were investigated thereby integrating three parameters, insulin release (RIA), 45Ca2+ uptake and 86Rb+ efflux (surrogate for K+ efflux) of INS-1 cells, an insulin secretory cell line. The presence of A1-receptors was demonstrated by Western blotting. The receptor nonselective adenosine analogue NECA (5-N-ethylcarboxyamidoadenosine) at high concentration (10 microM) had no effect on insulin release and 45Ca2+ uptake which could be interpreted as the sum of effects mediated by mutual antagonistic adenosine receptor subtypes. However, an inhibitory effect mediated by A1 receptor agonism was detected at 10 nM NECA and could be confirmed by adding the A1 receptor antagonist PSB-36 (1-butyl-8-(3-noradamantyl)-3-(3-hydroxy-propyl)xanthine). NECA inhibited 86Rb+ efflux which, however, did not fit with the simultaneous inhibition of insulin secretion. The selective A1 receptor agonist CHA (N6-cyclohexyladenosine) inhibited insulin release; the simultaneously increased Ca2+ uptake (nifedipine dependent) and inhibition of 86Rb+ efflux did not fit the insulin release data. The CHA effect (even the maximum effect at 50 microM) can be increased by 10 microM NECA indicating that CHA and NECA have nonspecific and physiologically non-relevant effects on 86Rb+ efflux in addition to their A1-receptor interaction. Since PSB-36 did not influence the NECA-induced inhibition of 86Rb+ efflux, the NECA effect is not mediated by potassium channel-linked A1 receptors. The nonselective adenosine receptor antagonist caffeine increased insulin release which was reversed by CHA as expected when hypothesizing that both act via A1 receptors in this case. In conclusion, stimulation of A1 receptors by receptor selective and nonselective compounds reduced insulin release which is not coupled to opening of potassium channels (86Rb+ efflux experiments) or inhibition of calcium channels (45Ca2+ uptake experiments). It may be expected that of all pleiotropic 2nd messengers, the cAMP system (not tested here) is predominant for A1 receptor effects and the channel systems (K+ and Ca2+) are of minor importance and do not contribute to insulin release though being coupled to the receptor in other tissues.

Variety of angiotensin receptors in 3T3-L1 preadipose cells and differentiated adipocytes.

A local paracrine acting angiotensin (ANG) system of preadipocytes and mature adipocytes is involved in metabolic effects and tissue differentiation. The present study reports on the investigation of binding affinities for various angiotensin receptors including their relevance in 3T3-L1 adipocytes and preadipocytes and 3T3-442A preadipocytes. Competitive binding studies using both 125I-ANG II and its more stable analogue 125I-SARILE for investigating AT1/AT2 binding sites in 3T3-L1 preadipocytes reveal a biphasic competition curve with KDs at a low and high nanomolar range. By using the AT2 receptor selective ligand 125I-CGP4112A the presence of high affinity AT2 binding sites in preadipocytes was observed. High nonspecific binding and a low receptor number is characteristic for all these experiments. An AT4 binding site (binding site for ANG IV) exists in 3T3-L1 and F442A preadipocytes and adipocytes with a high nanomolar KD. This low binding affinity was confirmed by a biological assay, the IRAP assay (=insulin regulated aminopeptidase assay). IRAP is associated with the AT4 receptor, which is a binding site at the luminal part of membrane bound IRAP. The curves for competition binding and for inhibition of IRAP activity are superimposable with respect to angiotensin IV. In conclusion, AT1 and AT2 binding sites are present in preadipocytes. AT2 receptor binding affinities are shown in preadipocytes for the first time. The description of a non-AT1/AT2 binding site with low affinity remains speculative albeit of high interest because antidiabetic and obesity related effects of angiotensin peptides and sartanes as antagonists are observed at these high concentrations. Local concentrations of ANG II and their degradation products may be extremely high. The low amounts of AT1 and AT2 binding sites emphasize the relevance of other binding sites in adipose tissue development and metabolic effects. The AT4 binding site seems to be one of the predominant receptors in adipose cells. Other degraded, but still bioactive peptides like ANG III, IV and ANG(1-7), activating receptors not influenced by ANG II, could be of importance.

Effect of two artichoke extracts (36_U and 36_EB) on rat ileum (with respect to bowel syndrome) and the peristaltic threshold.

The diagnosis and treatment of irritable bowel syndrome (IBS) are complicated. Artichoke extracts are well known to be helpful in various gastrointestinal disorders. A hydrophilic extract 36_U mainly containing luteolin-7-glycoside, luteolin-7-O-glucoside, small amounts of cynarin and luteolin increased contraction of rat ileum. This is mainly mediated by 5-HT(3) - and 5-HT(2) receptors but not 5-HT(4) receptors as can be derived by using specific antagonists such as tropisetrone, GR113806 and ketanserine. Additional mechanisms (receptors) are involved since the combination of these three antagonists was not able to fully prevent the contractive effect of extract 36_U. The lipophilic extract 36_EB mainly containing cynarin, luteolin including its glycosides, and cholorogenic acid in contrast to extract 36_U had a relaxing effect which could hardly be washed out. It was diminishing a serotonin effect and was not modified by ACh or substance P. The peristaltic threshold, i.e. the distension necessary for inducing a pathophysiologically relevant propulsion activity, is one of the important features being correlated with IBS. The peristaltic threshold was decreased by both serotonin and extract U_36. From the data it can be derived that the extract 36_U may be useful in IBS combined with obstipation when gastrointestinal contraction is necessary, whereas 36_EB may be useful in IBS combined with diarrhea when gastrointestinal relaxation is desired. Especially interesting are the influence on the threshold. It would be interesting to know which effects are mediated via cynarin and luteolin or its glycosides.

The effect of nigellone and thymoquinone on inhibiting trachea contraction and mucociliary clearance.

NIGELLA SATIVA L. has many effects including those on the gastrointestinal tract and trachea and is, therefore, used in the Mediteranean area and in India/Pakistan. Our aim was to investigate the effect of two main constituents, nigellone and thymoquinone, on trachea (antispasmodic effect) and their influence on respiratory clearance. The effects on Ba (2+)-, carbachol- and leukotriene-induced trachea contractions and the transport of the fluorescence dye rhodamin B concerning ciliary action in the tracheal area were investigated using a microdialysis technique. Nigellone and high concentrations of thymoquinone had a concentration-dependent inhibitory effect on the trachea when being contracted by the depolarizing effect of Ba (2+). The trachea contractions induced by leukotriene-d (4) were inhibited by nigellone and by thymoquinone. The cholinergic system (stimulation by carbachol) was hardly involved. The rate of ciliary clearance (mucociliary transport) was slightly modified by a high thymoquinone concentration (153.0 vs. 505.0 sec/12 mm distance, respectively), and was highly increased by nigellone (217.5 vs. 505.0 sec/12 mm distance). In conclusion, this study provides evidence for an antispasmodic effect and an increase in mucociliary clearance for nigellone but not for thymoquinone. Altogether the data indicate that nigellone but not thymoquinone may be useful in treatment of different respiratory diseases.

Large molecules as anti-adhesive compounds against pathogens.

Anti-adhesive compounds are potential prophylactic tools in alternative treatment regimes against bacterial infection, as bacterial adhesion is commonly mediated by carbohydrate-protein interactions between surface adhesions of microorganisms and the host cell. The use of exogenous polyvalent, high-molecular carbohydrates and tannin-like plant-derived compounds should antagonize the adhesive interaction. A range of carbohydrates and carbohydrate- and proanthocyanidin-enriched plant extracts were screened for potential anti-adhesive effects against Helicobacter pylori, Campylobacter jejuni, Porphyromonas gingivalis and Candida albicans in different in-situ assays on primary tissue. The adhesion of H. pylori on human stomach tissue was effectively blocked by glucuronic acid-enriched polysaccharides from immature okra fruits (Abelmoschus esculentus). These compounds also had strong in-vitro effects against C. jejuni (inhibition up to 80%), but were ineffective in an in-vivo study in infected chicken broilers due to metabolism in the gastrointestinal system. Polysaccharides from Glycyrrhizia glabra, also enriched with glucuronic acid, showed strong anti-adhesive properties against H. pylori and P. gingivalis (inhibition 60-70%). Pelargonium sidoides extract, containing mainly polymeric proanthocyanidins, was effective against H. pylori in a dose-dependent manner. Due to the multifunctional adhesive strategy of C. albicans, no effective compounds were detected against this yeast. Structure-activity relationships are presented and the potential in-vivo use of carbohydrate-based anti-adhesives is discussed.

The effect of thyme extract on beta2-receptors and mucociliary clearance.

UNLABELLED: Thyme is a broncholytic und secretomotoric agent. Thus, our aim was to investigate the influence of a thyme extract on beta (2)-receptors in competition binding experiments and relaxation experiments on rat uteri and trachea. Furthermore, the influence of the extract on respiratory clearance was of interest. Binding experiments were performed using purified rat lung membranes with the beta(2)-receptor ligand [(125)I]-CYP {[(125)I]-(+/-)-Iodocyanopindolol}. The transport of the fluorescence dye rhodamin 123 concerning ciliary action in the tracheal area of a mouse was investigated using a microdialysis technique. The thyme extract reduces only slightly [(125)I]-CYP binding and amplifies the displacement of [(125)I]-CYP by propranolol (non-specific beta-receptor antagonist): the displacement curve in the concentration range representing beta (2)-receptors (nM) is shifted to the left. Thyme extract had relaxing effects on organs possessing beta (2)-receptors (uterus and trachea). The propranolol-induced antagonism to isoprenaline is reverted concentration-dependently by the extract. A duplication of the rate of ciliary clearance by the extract was observed. IN CONCLUSION: 1) There is evidence for an influence of a thyme extract on beta (2)-receptors by both binding studies and biological effects: As can be derived from the shift of the propranolol displacement curve (nM), ingredients of the thyme extract slightly interact with beta (2)-receptors in rat lung tissue. This effect is indirect since no full range competition curve was reached. 2) An at least indirect interaction with beta (2)-receptors in rat uteri and trachea is revealed by a decreased antagonism of propranolol on the relaxing effect of isoprenaline by the plant extract. 3) An additional mechanism is presumed because at high extract concentrations isoprenaline-induced relaxation is complete, whereas the displacement of propranolol at beta (2)-receptors is only weak. 4) Thyme extract has an indirect (modulatory) effect on the beta (2)-receptor system. 5) Mucociliary clearance is improved in vivo. Its mechanism has still to be elucidated.

The effect of the volatile oil from ginger rhizomes (Zingiber officinale), its fractions and isolated compounds on the 5-HT3 receptor complex and the serotoninergic system of the rat ileum.

A contribution of the volatile oil from ginger rhizomes (Zingiber officinale Roscoe) on inhabiting the 5-HT3 receptor complex had been shown. In the present study a possible interaction of some compounds of the volatile oil with the 5-HT3 receptor system expressed in N1E-115 cells and with the serotoninergic system of the rat ileum was investigated. The volatile oil was obtained by steam distillation and fractionated using a silica gel column resulting in five fractions. Compounds of the fractions were identified by GC-MS. The influence of the volatile oil, its fractions and pure components on serotonin-induced [14C]guanidinium influx into N1E-115 cells was measured indicating the inhibitory interaction with the 5-HT3 receptor channel system. Most potent inhibitors of cation influx were the volatile oil, fraction 4, beta-pinene, terpinolene, alpha-copaene and alpha-phellandrene. The volatile oil and fractions 1 and 4 were not able to significantly influence either serotonin (10 microM)-induced maximum contraction of the rat ileum or the second phase of the biphasic contraction 2.5 min after serotonin addition. However, beta-pinene, terpinolene and alpha-phellandrene reduced both contractions. In conclusion, the volatile oil and distinct compounds such as terpinolene, beta-pinene and alpha-phellandrene interact with 5-HT3 receptor channel system and possess an antispasmodic effect at the rat ileum.

Hypoglycaemic constituents of Stachytarpheta cayennensis leaf.

The aqueous infusion (tea) of Stachytarpheta cayennensis leaves is used ethnomedically in Peru, Nigeria and other tropical countries for the management of diabetes. Oral administration (p. o.) of aqueous (125 mg/kg) and methanolic (2000 mg/kg) extracts of the leaves to alloxan-diabetic rats showed significant blood glucose reductions by 43 and 53%, respectively, at the end of a 4 hour period similar to the strong effect of glibenclamide (5 mg/kg, P. O.). The methanolic extract was successively partitioned into ethyl acetate, butanol and water fractions, and the same test showed that the butanol fraction (2000 mg/kg) had the highest (50%) hypoglycaemic activity at 4 hours after oral administration. It was also the most active fraction when tested in vitro [insulin release from an insulin secreting cell line (INS-1)] and was also active in normal rats and rats made hyperglycaemic by a glucose load. Its activity was comparable to that of glibenclamide (positive control) in these models. This active butanol fraction was subjected to chromatographic subfractionation; some subfractions reduced hyperglycaemia in alloxan-diabetic rats to 60 and 78% and induced insulin release from the INS-1 cells; other subfractions, however, gave hyperglycaemic activities IN VIVO and inhibition of insulin release from the INS-1 cells. Three major compounds of the butanol fraction were isolated and characterised as 6beta-hydroxyipolamide, ipolamide and isoverbascoside; they increased insulin secretion from INS-1 cells to 125, 128 and 127%, respectively, whereas glibenclamide increased insulin secretion to 157%. The results justify the ethnomedical use of the plant in the management of diabetes and suggest that the butanol fraction and some of its isolated constituents mediate their actions primarily by stimulating insulin release directly.

Antispasmodic activity of an extract from Plantago lanceolata L. and some isolated compounds.

An ethanolic spissum extract of the aerial parts of Plantago lanceolata L. was examined for antispasmodic activity on isolated ileum and trachea of the guinea-pig. Isolated constituents were investigated as well. The P. lanceolata extract inhibited the contractions of the guinea-pig ileum that were induced by various agonists such as acetylcholine (ACh), histamine, potassium and barium ions. Additionally the trachea contractions induced by barium ions were inhibited. The compounds luteolin, acteoside, plantamajoside an catalpol peracetate but not catalpol, isoacteoside, lavandulifolioside and aucubin inhibited the ACh-induced contractions of the guinea-pig ileum. Luteolin and acteoside reduced the barium-induced contractions of the guinea-pig trachea. Two recently isolated compounds did not show antispasmodic activity: luteolin-3',7-diglucuronide and beta-hydroxy-acteoside.

The impact of adenosine and A(2B) receptors on glucose homoeostasis.

Adenosine and adenosine receptor antagonists are involved in glucose homoeostasis. The participating receptors are not known, mainly due to a lack of specific agonists and antagonists, but are reasonable targets for anti-diabetic therapy. The stable, albeit nonselective, adenosine analogue NECA (5'-N-ethylcarboxamidoadenosine) (10 microM) reduced glucose-stimulated insulin release from INS-1 cells. This was mimicked by A(1)-(CHA), A(2A)-(CGS-21680) and A(3)-receptor agonists (Cl-IB-MECA). Two newly synthesized A(2B)-receptor antagonists, PSB-53 and PSB-1115, counteracted the inhibitory effect of NECA. These in-vitro effects were mirrored by in-vivo data with respect to CHA, CGS and Cl-IB-MECA. Distinct concentrations of either PSB-53 or PSB-1115 reversed the decrease in plasma insulin induced by NECA. This was not mimicked by a corresponding change in blood glucose. The effect of PSB-1115 was also obvious in diabetic GotoKakizaki rats: plasma insulin was increased whereas blood glucose was unchanged. During most experiments the effects on blood glucose were not impressive probably because of the physiologically necessary homoeostasis. The adenosine levels were not different in normal Wistar rats and in diabetic GotoKakzaki rats. Altogether the A(2B)-receptor antagonists showed an anti-diabetic potential mainly by increasing plasma insulin levels under conditions when the adenosine tonus was elevated in-vivo and increased insulin release in-vitro.

5-HT3 receptor blocking activity of arylalkanes isolated from the rhizome of Zingiber officinale.

Different extracts (ethanolic, hexane, aqueous) of ginger (rhizomes of Zingiber officinale) and the essential oil were tested using [14C]guanidinium influx into N1E-115 cells and the isolated rat ileum in order to identify their activity in inhibiting 5-HT3 receptor function. The hexane extract proved to be the most active and yielded upon bioassay-guided fractionation nine constituents: [6]-, [8]-, [10]-gingerols, [6]- and [8]-shogaols which were previously shown as active in vivo against cytotoxic drug-induced emesis; [4]-gingerol, [6]-gingerdiol, diacetyl-[6]-gingerdiol and [6]-dehydrogingerdione have not been previously tested for anti-emetic or 5-HT3 receptor antagonistic effects. Even though the latter four compounds are only minor constituents, their identification contributed towards the characterisation of a structure-activity relationship of this class of compounds. The order of potency for the nine constituents in the N1E-115 cell system was [6]-gingerdiol approximately diacetyl-[6]-gingerdiol approximately [6]-dehydrogingerdione approximately [6]-shogaol > or = [8]-shogaol approximately [8]-gingerol > [10]-gingerol > or = [6]-gingerol > [4]-gingerol.

Heterologous desensitization of insulin secretion by GIP (glucose-dependent insulinotropic peptide) in INS-1 cells: the significance of Galphai2 and investigations on the mechanism involved.

Heterologous desensitization is a term that describes the observation that chronic exposure of a cell to an agonist attenuates its response to other agonists. To characterize the cellular mechanisms that might be responsible for heterologous desensitization in an insulin secretory cell system (INS-1), we investigated the link between G-protein alphai2 level and insulin secretion as the biological effect after prolonged incubation with glucose-dependent insulinotropic polypeptide (GIP). Persistent activation (8 h) of the GIP signalling pathway decreased the GLP (glucagon-like peptide)-1 dependent insulin secretion (specific radioimmunoassay) accompanied by an upregulation of G-protein alphai2 protein level to about 126% whereas G-protein alphai3 and alphas protein levels remained unchanged (assessed by Western blots using specific antibodies). This was accompanied by similar changes in Galphai2 mRNA. By using either the CaM kinase II inhibitor KN-62, the calcineurin inhibitor FK 506 or the protein kinase A (PKA) inhibitor Rp-8-Br-cAMPS, the GIP-mediated Galphai2 mRNA increase was fully reversed. Heterologous desensitization of GLP-1-dependent insulin secretion by pretreatment with GIP, however, was not inhibited by calcium/calmodulin-dependent enzymes (using KN-62 and FK 506), but only by suppressing the cAMP/PKA signalling pathway using Rp-8-Br-cAMPS. The outcome is not disturbed by effects initiated by these compounds per se since an 8-h preincubation of cells did not affect glucose-induced insulin secretion. We, therefore, suggest that heterologous desensitization in INS-1 cells may be mediated by Galphai2 changes but depend on the cAMP/PKA signalling pathway probably distant form the Galphai2 protein.

Influence of diadenosine tetraphosphate (Ap4A) on lipid metabolism.

Diadenosine polyphosphates (Ap(x)A) are physiologically released and may be partly involved in the pathogenesis of diabetes mellitus. Ap(4)A (diadenosine tetraphosphate) leads to an increase in blood glucose while it decreases insulin levels in plasma. A possible link between Ap(x)A and diabetes mellitus-associated diseases such as insulin resistance and hyperlipidemia (plasma free fatty acids, cholesterol and its biosynthesis, triacylglycerols) has not been investigated yet. Parameters such as free fatty acid and cholesterol content in blood were determined enzymically. The biosynthesis of cholesterol and triacylglycerols was determined in HepG2 cells using the radioactive precursor [(14)C]-acetate and by using gas chromatography. Plasma free fatty acids were significantly decreased 5 and 10 min after an Ap(4)A bolus (0.75 mg kg(-1) b.w.) given to rats. Plasma cholesterol was reduced 5 and 60 min after Ap(4)A administration. LPDS (lipoprotein-deficient serum)-stimulated cholesterol biosynthesis in HepG2 cells was significantly reduced after 1 h incubation with Ap(4)A. Triacylglycerol (TAG) biosynthesis in HepG2 cells was not significantly influenced by Ap(4)A; there was just a tendency for a concentration-dependent decrease in TAG levels. In conclusion Ap(4)A as a diabetogenetic compound is not likely to be responsible for the development of insulin resistance or of hyperlipidemia. Parameters such as free fatty acids, cholesterol and triacylglycerols are not elevated by Ap(4)A, but are even decreased. Ap(4)A seems to be involved in the development of diabetes mellitus by increasing blood glucose and decreasing plasma insulin as shown earlier, but not in diabetes mellitus-associated diseases such as insulin resistance or hyperlipidemia.

Vascular smooth muscle cells (VSMC) proliferation of streptozotocin-diabetic animals induced by diadenosine polyphosphates.

UNLABELLED: Specific binding sites for diadenosine polyphosphates (Ap (4)A, Ap (5)A, Ap (6)A) exist in VSMC (cultured vascular smooth muscle cells). These compounds may regulate VSMC growth and proliferation which is a key event in atherogenesis. Since diabetes is a known risk factor for atherosclerosis, the proliferation of VSMC from normoglycemic (control) and hyperglycemic (diabetic) rats were compared and the possibly involved receptors for diadenosine polyphosphates inducing this effect were investigated. Diabetes was induced by streptozotocin (66 mg/kg i.p.) and VSMC were prepared from rat aorta (primary culture). ( (3)H)thymidine incorporation was a measure of cell proliferation. For all diadenosine polyphosphates tested a stimulatory effect was observed as a bell-shaped concentration-response curve and a maximum effect at 10 micro M (physiological concentration). Ap (6)A has the most prominent effect (247.8 +/- 33.2 % increase over basal). In VSMC of diabetic rats the effects were even more prominent (Ap (5)A: 430.1 +/- 62.7 %). ATP (a degradation product of Ap (6)A) is able to increase the maximum effect of 10 micro M Ap (6)A. UTP (P2Y (2) agonist) exhibits a weaker proliferation. 1 micro M suramin (P2 receptor antagonist) shifts the concentration response curve of ATP and of Ap (6)A to the right. In contrast, 10 micro M PPADS (P2 X receptor antagonist) has no effect. There is no difference between VSMC of normal and diabetic rats in this respect. ADP, AMP, and adenosine exhibit a dual proliferative effect. The effect of either of these 3 compounds is much higher in VSMC of diabetic rats than of controls. 2MeSATP (P2Y (1) agonist) and alpha,beta-Methylen-ATP (P2X agonist) were not effective in VSMC of both normoglycemic and diabetic rats. IN CONCLUSION: The proliferative effect of diadenosine polyphosphates and some degradation products is more pronounced in VSMC of diabetic than of normal rats. Ap (6)A acts maximally by itself and not by its degradation product ATP. Adenosine receptors or an unknown P2Y (ApxA) receptor may be involved in proliferative effects, but not P2X and P2Y (1) receptors irrespective of a diabetic situation.

The serpins alpha-1-antitrypsin and alpha-1-antichymotrypsin specifically interact with immunophilins.

In a yeast two-hybrid screen FKBP13, a member of the FK506 Binding Protein (FKBP) family, was detected to interact with the serpin alpha-1-antichymotrypsin (ACT). The specificity of the interaction was confirmed in vitro and by the lack of interaction of ACT with FKBP25 and FKBP52. Mutational analysis of ACT revealed that the entire protein is necessary to interact with FKBP13. ACT but also different unrelated small regions of the ACT protein were able to interact with the smaller FKBP12, demonstrating a rather nonspecific interaction with this immunophilin. Naturally occuring mutants of ACT were able to interact as well. Antitrypsin (AT) closely related to ACT did only interfere with FKBP12 a protein that does presumably not reside in the same cellular compartment with AT and ACT. Both serpins interacted with the unrelated immunophilin cyclophilin A. In conclusion the serpin alpha-1-antichymotrypsin physiologically interacts with the ER-immunophilin FKBP13 and the secreted immunophilin cyclophilin A in vivo whereas alpha-1-antitrypsin might only react with cyclophilin A; both serpins may be controlled thereby in their genuine function.

Diadenosine tetraphosphate (Ap4A) induces a diabetogenic situation: its impact on blood glucose, plasma insulin, gluconeogenesis, glucose uptake and GLUT-4 transporters.

Diadenosine polyphosphates such as Ap4A are physiologically released compounds for which both receptors as well as a role as second messengers for influencing insulin release have been shown. So far little is known about their pathophysiological impact on diabetes with respect to blood glucose and plasma insulin, glucose production via gluconeogenesis, glucose uptake and GLUT-4 expression. Rats given an intravenous bolus of Ap4A (0.75 mg/kg) developed a rapid and dramatic increase in blood glucose. Plasma insulin was only transiently increased (for 4 min), but did not follow the normally stimulatory effect of the elevated blood glucose. A bolus of 25 microg Ap4A quickly increased glucose release from perfused rat liver. Glucose uptake was reduced in 3T3 adipocytes. Reduced amounts of translocated GLUT-4 were found in 3T3 cell membranes incubated with 10 microM Ap4A. Thus, Ap4A itself induces a diabetic situation which is likely to be mediated by an increase in gluconeogenesis and/or an insulin resistance caused by a decrease in GLUT-4 and an attenuation of glucose uptake.