alpha2A-adrenoceptor antagonism increases insulin secretion and synergistically augments the insulinotropic effect of glibenclamide in mice

BACKGROUND AND PURPOSE: The imidazoline-type alpha2-adrenoceptor antagonists (+/-)-efaroxan and phentolamine increase insulin secretion and reduce blood glucose levels. It is not known whether they act by antagonizing pancreatic beta-cell alpha2-adrenoceptors or by alpha2-adrenoceptor-independent mechanisms. Many imidazolines inhibit the pancreatic beta-cell KATP channel, which is the molecular target of sulphonylurea drugs used in the treatment of type II diabetes. To investigate the mechanisms of action of (+/-)-efaroxan and phentolamine, alpha2A-adrenoceptor knockout (alpha2A-KO) mice were used. EXPERIMENTAL APPROACH: Effects of (+/-)-efaroxan, 5 mg kg(-1), and phentolamine, 1 mg kg(-1), on blood glucose and insulin levels were compared with those of the non-imidazoline alpha2-adrenoceptor antagonist [8aR,12aS,13aS]-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine (RS79948-197), 1 mg kg(-1), and the sulphonylurea glibenclamide, in alpha2A-KO and control (wild type (WT)) mice.Key results:In fed WT mice, (+/-)-efaroxan, phentolamine and RS79948-197 reduced blood glucose and increased insulin levels. Fasting abolished these effects. In fed alpha2A-KO mice, (+/-)-efaroxan, phentolamine and RS79948-197 did not alter blood glucose or insulin levels, and in fasted alpha2A-KO mice, blood glucose levels were increased. Glibenclamide, at a dose only moderately efficacious in WT mice (5 mg kg(-1)), caused severe hyperinsulinaemia and hypoglycaemia in alpha2A-KO mice. This was mimicked in WT mice by co-administration of RS79948-197 with glibenclamide. CONCLUSIONS AND IMPLICATIONS: These results suggest that (+/-)-efaroxan and phentolamine increase insulin secretion by inhibition of beta-cell alpha2A-adrenoceptors, and demonstrate a critical role for alpha2A-adrenoceptors in limiting sulphonylurea-induced hyperinsulinaemia and hypoglycaemia.     

Effects of sulphonylureas on the volume-sensitive anion channel in rat pancreatic β-cells

1. The volume-sensitive anion conductance in rat pancreatic β-cells was studied directly using the conventional whole-cell and perforated patch recording techniques, and indirectly by measuring ³H-taurine efflux from pre-loaded, perifused islets.
2. Using the conventional whole-cell recording configuration, activation of the outwardly-rectifying, DIDS-sensitive conductance was induced by glibenclamide (10 μM) but not by tolbutamide (100 μM) nor by meglitinide (20 μM). A high concentration of glibenclamide (100 μM) caused a voltage-and time-dependent inhibition of the conductance. Tolbutamide had a modest inhibitory effect on swelling-induced inward currents.
3. In perforated patch recordings, glibenclamide, tolbutamide and meglitinide were all without effect on the conductance, although activation could be induced under these conditions by exposure to a hypotonic bath solution.
4. The rate of efflux of ³H-taurine, a marker for activity of the volume-sensitive anion channel, from preloaded, perifused islets was markedly stimulated by exposure to a hypotonic solution. However, glibenclamide and tolbutamide were both without effect.
5. Electrical activity of β-cells in response to glibenclamide or tolbutamide was not inhibited by 4,4′-dithiocyanatostilbene-2,2′-disulphonic acid (DIDS), an inhibitor of the volume-sensitive anion channel.
6. It is concluded that activity of the volume-sensitive anion conductance in rat pancreatic β-cells is not modulated by the sulphonylurea receptor. The activation of the conductance by glibenclamide in whole-cell recordings could be the result of a non-specific interaction of the drug with plasma membrane lipids.

     

Glycogen synthase kinase 3: more than a namesake

Glycogen synthase kinase 3 (GSK3), a constitutively acting multi-functional serine threonine kinase is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. These diverse multiple functions attributed to GSK3 can be explained by variety of substrates like glycogen synthase, τ protein and β catenin that are phosphorylated leading to their inactivation. GSK3 has been implicated in various diseases such as diabetes, inflammation, cancer, Alzheimer''s and bipolar disorder. GSK3 negatively regulates insulin-mediated glycogen synthesis and glucose homeostasis, and increased expression and activity of GSK3 has been reported in type II diabetics and obese animal models. Consequently, inhibitors of GSK3 have been demonstrated to have anti-diabetic effects in vitro and in animal models. However, inhibition of GSK3 poses a challenge as achieving selectivity of an over achieving kinase involved in various pathways with multiple substrates may lead to side effects and toxicity. The primary concern is developing inhibitors of GSK3 that are anti-diabetic but do not lead to up-regulation of oncogenes. The focus of this review is the recent advances and the challenges surrounding GSK3 as an anti-diabetic therapeutic target.British Journal of Pharmacology (2009) doi:10.1111/j.1476-5381.2008.00085.x     

Differential Gene Expression in GPR40-Overexpressing Pancreatic β-cells Treated with Linoleic Acid

"G protein-coupled receptor 40" (GPR40), a receptor for long-chain fatty acids, mediates the stimulation of glucose-induced insulin secretion. We examined the profiles of differential gene expression in GPR40-activated cells treated with linoleic acid, and finally predicted the integral pathways of the cellular mechanism of GPR40-mediated insulinotropic effects. After constructing a GPR40-overexpressing stable cell line (RIN-40) from the rat pancreatic β-cell line RIN-5f, we determined the gene expression profiles of RIN-5f and RIN-40. In total, 1004 genes, the expression of which was altered at least twofold, were selected in RIN-5f versus RIN-40. Moreover, the differential genetic profiles were investigated in RIN-40 cells treated with 30 µM linoleic acid, which resulted in selection of 93 genes in RIN-40 versus RIN-40 treated with linoleic acid. Based on the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG, http://www.genome.jp/kegg/), sets of genes induced differentially by treatment with linoleic acid in RIN-40 cells were found to be related to mitogen-activated protein (MAP) kinase- and neuroactive ligand-receptor interaction pathways. A gene ontology (GO) study revealed that more than 30% of the genes were associated with signal transduction and cell proliferation. Thus, this study elucidated a gene expression pattern relevant to the signal pathways that are regulated by GPR40 activation during the acute period. Together, these findings increase our mechanistic understanding of endogenous molecules associated with GPR40 function, and provide information useful for identification of a target for the management of type 2 diabetes mellitus.     

Regulation by tolbutamide and diazoxide of the electrical activity in mouse pancreatic β-cells recorded in vivo

1. The glucose-dependence of β-cell electrical activity and the effects of tolbutamide and diazoxide were studied in anaesthetized mice.
2. In untreated animals there was a direct relationship between glycaemia and the burst pattern of electrical activity. Animals with high glucose concentration showed continuous electrical activity. The application of insulin led to a steady decrease in blood glucose concentration and a transition from continuous to oscillatory activity at 7.7±0.1 mM glucose (mean±s.d.) and a subsequent transition from oscillatory to silent at 4.7±0.6 mM glucose.
3. At physiological blood glucose concentrations the electrical activity was oscillatory. The injection of tolbutamide (1800 mg kg⁻¹) transformed this oscillatory pattern into one of continuous electrical activity. The increased electrical activity was associated with a decrease in blood glucose concentration from 7.1±0.9 (control) to 5.5±1.0 mM (10 min after tolbutamide injection). The effects of tolbutamide are consistent with a direct blocking effect on the K_ATP channel that leads to membrane depolarization.
4. The injection of diazoxide (6000 mg kg⁻¹) hyperpolarized the cells and transformed the oscillatory pattern into a silent one. This is consistent with a direct stimulant effect by diazoxide on the K_ATP channel. The use of tolbutamide or diazoxide correspondingly led to the lengthening or shortening of the active phase of electrical activity, respectively. This indicates that in vivo, such activity can be modulated by the relative degree of activation or inhibition of the K_ATP channel.
5. These results indicate that under physiological conditions, tolbutamide and diazoxide have direct and opposite effects on the electrical activity of pancreatic β-cells, most likely through their action on K_ATP channels. This is consistent with previous work carried out on in vitro models and explains the drugs hypo- and hyperglycaemic effects.

     

Effect of PRX-1 Downregulation in the Type 1 Diabetes Microenvironment

Type 1 diabetes (T1D) is caused by dysregulation of the immune system in the pancreatic islets, which eventually leads to insulin-producing pancreatic β-cell death and destabilization of glucose homeostasis. One of the major characteristics of T1D pathogenesis is the production of inflammatory mediators by macrophages that result in destruction or damage of pancreatic β-cells. In this study the inflammatory microenvironment of T1D was simulated with RAW264.7 cells and MIN6 cells, acting as macrophages and pancreatic β-cells respectably. In this setting, peroxiredoxin-1, an anti-oxidant enzyme was knocked down to observe its functions in the pathogenesis of T1D. RAW264.7 cells were primed with lipopolysaccharide and co-cultured with MIN6 cells while PRX-1 was knocked down in one or both cell types. Our results suggest that hindrance of PRX-1 activity or the deficiency of this enzyme in inflammatory conditions negatively affects pancreatic β-cell survival. The observed decrease in viability of MIN6 cells seems to be caused by nitric oxide production. Additionally, it seems that PRX-1 affects previously reported protective activity of IL-6 in pancreatic β cells as well. These results signify new, undiscovered roles for PRX-1 in inflammatory conditions and may contribute toward our understanding of autoimmunity.     

Association of serum 25-hydroxyvitamin D with insulin resistance and β-cell function in a healthy Chinese female population

Aim:

To assess associations of the serum level of 25-hydroxyvitamin D with insulin resistance and β-cell function in a healthy Chinese female population.

Methods:

This cross-sectional study included 1382 female participants free of type 2 diabetes who were recruited in Shanghai. Blood samples were collected within a winter season and the serum levels of 25-hydroxyvitamin D, fasting plasma glucose and insulin, and other biochemical parameters were determined. Insulin resistance and β-cell function were assessed using the homeostasis model assessments of insulin resistance (HOMA-IR) and β-cell function (HOMA-B), respectively.

Results:

Multiple linear regression analyses adjusted for age, parathyroid hormone, Ca²⁺ and BMI revealed that independent inverse associations existed between the serum level of 25-hydroxyvitamin D and HOMA-IR (P<0.001) and between the serum level of 25-hydroxyvitamin D and HOMA-B (P=0.001).

Conclusion:

Serum vitamin D level is significantly and independently associated with insulin resistance and β-cell function in a healthy Chinese female population.     

Modulatory role of 1,25 dihydroxyvitamin D3 on pancreatic islet insulin release via the cyclic AMP pathway in the rat

1. Previous studies have shown that vitamin D₃ deficiency impairs the insulin response to glucose via an alteration of signal transduction pathways, such as Ca²⁺ handling and the phosphoinositide pathway. In the present study the adenylyl cyclase pathway was examined in islets from 3 independent groups: normal rats, 4 weeks-vitamin D₃ deficient rats and one week-1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) treated rats.
2. We found that the very low rate of insulin release observed in vitamin D₃ deficient rats could be restored in vitamin D₃ deficient islets only with high concentrations of dioctanoyl-cyclic AMP (DO-cyclic AMP), whereas 1,25(OH)₂D₃ improved the sensitivity of the islets to this exogenous cyclic AMP analogue.
3. The beneficial effect of 1,25(OH)₂D₃ observed with or without DO-cyclic AMP was protein kinase A-dependent, since the addition of N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulphonamide (H-89), a specific inhibitor of cyclic AMP-dependent protein kinases, decreased the insulin release of treated rats back to the level seen in vitamin D₃ deficient islets.
4. The low rate of insulin release could not be consistently related to an alteration in cyclic AMP content of the islets. Indeed, low insulin response to a barium + theophylline stimulus observed in vitamin D₃ deficient islets was paradoxically associated with a supranormal cyclic AMP content in the islets.
5. This paradoxical increase in cyclic AMP observed in these conditions could not be attributed to a lower total phosphodiesterase (PDE) activity, although the portion of Ca²⁺-calmodulin-independent PDE was predominant in islets from vitamin D₃ deficient rats.
6. On the other hand, the higher cyclic AMP content of vitamin D₃ deficient islets could be related to an increase in glucagon-induced cyclic AMP synthesis in relation to the hyperglucagonaemia previously observed in vitamin D₃ deficient rats. Since higher concentrations of exogenous glucagon and higher endogenous cyclic AMP concentrations were required in vitro to restore insulin release to normal values, the cyclic AMP-dependent pathways that usually potentiate insulin secretion appeared to be less efficient in relation to an alteration in the post cyclic AMP effector system.
7. 1,25(OH)₂D₃ exerted a stimulating effect on insulin release via protein kinase A activation but reduced the supranormal cyclic AMP synthesis, thus exerting a differential modulatory influence on biochemical disturbances in islets induced by vitamin D₃ deficiency.

     

How biologics targeting the IL-1 system are being considered for the treatment of type 2 diabetes

Metabolic diseases are associated with activation of the innate immune system in various tissues and characterized by elevated inflammatory factors and the presence of immune cells. Type 2 diabetes develops when islet beta cells are deficient in producing sufficient insulin to overcome peripheral insulin resistance. Intra-islet IL-1β activity diminishes beta cell function and survival and governs islet inflammation. Targeting the IL-1 system with the IL-1 receptor antagonist IL1Ra improved insulin secretion, glycaemia and reduced systemic inflammation in a proof of concept study with patients with type 2 diabetes. Currently, long lasting and specific IL-1β blocking antibodies are being evaluated in clinical trials and this may lead to a novel cytokine-based treatment for type 2 diabetes.     

The ability of a new hypoglycaemic agent,A-4166, compared to sulphonylureas,to increase cytosolic Ca2+ in pancreatic β-cells under metabolic inhibition

1. N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (A-4166) is a new non-sulphonylurea oral hypoglycaemic agent which stimulates insulin release by increasing cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in β-cells.
2. We studied comparative effects of A-4166 and sulphonylureas on [Ca²⁺]_i, measured by dual-wavelength fura-2 microfluorometry, in single rat pancreatic β-cells under normal conditions and conditions where glucose metabolism was inhibited.
3. A glucokinase inhibitor, mannoheptulose (10 mM), a mitochondrial respiratory inhibitor, KCN (100 μM), and uncouplers, dinitrophenol (DNP, 50 μM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 0.3 μM), were used to abolish glucose-induced increases in [Ca²⁺]_i in a reversible manner.
4. Under control conditions, A-4166 was one order more potent than tolbutamide in increasing [Ca²⁺]_i, and maximal responses were evoked by 30 μM A-4166 and 300 μM tolbutamide. These equipotent concentrations were employed for the comparative study where glucose metabolism was inhibited.
5. In the presence of mannoheptulose, [Ca²⁺]_i responses to tolbutamide, but not those to A-4166, were attenuated in a reversible manner.
6. KCN, DNP and FCCP inhibited [Ca²⁺]_i responses to tolbutamide to a much greater extent than those to A-4166. Responses to tolbutamide even at 3.3 times the equipotent concentration (1000 μM) were also markedly attenuated by these inhibitors. Responses evoked by another sulphonylurea, gliclazide, were inhibited by DNP to a larger extent than A-4166-induced responses.
7. The results indicate that A-4166 acts more effectively than sulphonylureas to increase [Ca²⁺]_i in β-cells during metabolic inhibition.

     

Biomarkers of Exposure to Zearalenone in In Vivo and In Vitro Studies

The measurement of human exposure to mycotoxins is necessary for its association with adverse health effects. This exposure is usually estimated from contamination levels of foodstuffs, which are the primary source of toxin exposure, and data on food consumption patterns. However, variations in contamination level, intestinal absorption, toxin distribution, and excretion lead to individual variations in toxin exposure that can be more readily measured with a biomarker. This review deals with the latest literature information about ZEN biomarkers in humans, animals, and cell line cultures. Their presence in urine, biomarkers that have effects in the kidney, liver, reproductive system and blood and biomarkers of cell response have been reported. It has highlighted the importance of determining α-zearalenol and β-zearalenol biomarkers to estimate the probable dietary intake (PDI) of a specific population or to characterize the severity of exposure to ZEN in animals or cell lines. α-ZEL and β-ZEL are cytotoxic by inhibiting cell proliferation, total protein and DNA syntheses, in this sense, an induction of expression proteins Hsp27 and Hsp70 was observed, and an increase in gene expression (TLR4, NF-kBp65, TNF-α, IL-1β, IL-6, IL-8, MGMT, α-GST, Hsp70, Nrf2, L-Fabp, HO-1, MAPK8), the determination of which indicates an oxidative stress effect. The integrity of the cell or tissue membrane is assessed by lactate dehydrogenase (LDH), which increase at exposure of ZEN (84.2 µM), and the proportions of some fatty acids of the renal tissue membrane were increased at treatments with ZEN. This review allows starting future studies of animal and population exposure in parallel with those of health effects works.     

Effect of long- and short-term treatments with pravastatin on diabetes mellitus and pancreatic fibrosis in the Otsuka–Long–Evans–Tokushima Fatty rat

Background and purpose:

The effects of statins on diabetes mellitus (DM) are controversial, and their effects on pancreatic fibrosis are poorly defined. We investigated the effect of long- and short-term treatments with pravastatin on the development of DM and pancreatic fibrosis in DM-prone Otsuka–Long–Evans–Tokushima Fatty (OLETF) rats.

Experimental approach:

Male OLETF rats were divided into four groups at 12 weeks of age. The first group received a standard rat diet until the end of the experimental period at age 80 weeks. The second group was given a diet containing 0.05% pravastatin from 12 weeks of age, before the onset of DM and pancreatic fibrosis, and the third group was given the same pravastatin diet from 28 weeks of age, after the onset of DM and pancreatic fibrosis, until age 80 weeks. The fourth group received the same pravastatin diet only for 16 weeks, from 12 to 28 weeks of age, and switched to a standard diet. Progressions of DM and pancreatic fibrosis were evaluated.

Key results:

Long-term treatments with pravastatin, either from 12 or 28 weeks of age, decreased serum glucose concentration and fibrotic area, elevated superoxide dismutase activity and down-regulated transforming growth factor-β1 mRNA in the pancreas. In contrast, after a short-term treatment with pravastatin, these parameters markedly deteriorated after its cessation.

Conclusions and implications:

The results suggest that long-term treatment with pravastatin improves DM and pancreatic fibrosis via anti-oxidative and anti-fibrotic properties, whereas cessation of pravastatin abolishes these beneficial effects, and accelerates DM and pancreatic fibrosis.     

Interaction of N-benzoyl-D-phenylalanine and related compounds with the sulphonylurea receptor of β-cells

1. The structure activity relationships for the insulin secretagogues N-benzoyl-D-phenylalanine (NBDP) and related compounds were examined at the sulphonylurea receptor level by use of cultured HIT-T15 and mouse pancreatic β-cells. The affinities of these compounds for the sulphonylurea receptor were compared with their potencies for K_ATP-channel inhibition. In addition, the effects of cytosolic nucleotides on K_ATP-channel inhibition by NBDP were investigated.
2. NBDP displayed a dissociation constant for binding to the sulphonylurea receptor (K_D value) of 11 μM and half-maximally effective concentrations of K_ATP-channel inhibition (EC₅₀ values) between 2 and 4 μM (in the absence of cytosolic nucleotides or presence of 0.1 mM GDP or 1 mM ADP).
3. In the absence of cytosolic nucleotides or presence of GDP (0.1 mM) maximally effective concentrations of NBDP (0.1–1 mM) reduced K_ATP-channel activity to 47% and 44% of control, respectively. In the presence of ADP (1 mM), K_ATP-channel activity was completely suppressed by 0.1 mM NBDP.
4. The L-isomer of N-benzoyl-phenylalanine displayed a 20 fold lower affinity and an 80 fold lower potency than the D-isomer.
5. Introduction of a p-nitro substituent in the D-phenylalanine moiety of NBDP did not decrease lipophilicity but lowered affinity and potency by more than 30 fold.
6. Introduction of a p-amino substituent in the D-phenylalanine moiety of NBDP (N-benzoyl-p-amino-D-phenylalanine, NBADP) reduced lipophilicity and lowered affinity and potency by about 10 fold. This loss of affinity and potency was compensated for by formation of the phenylpropionic acid derivative of NBADP. A similar difference in affinity was observed for the sulphonylurea carbutamide and its phenylpropionic acid derivative.
7. Replacing the benzene ring in the D-phenylalanine moiety of NBDP by a cyclohexyl ring increased lipophilicity, and the K_D and EC₅₀ values were slightly lower than for NBDP. Exchange of both benzene rings in NBDP by cyclohexyl rings further increased lipophilicity without altering affinity and potency.
8. This study shows that N-acylphenylalanines interact with the sulphonylurea receptor of pancreatic β-cells in a stereospecific manner. Their potency depends on lipophilic but not aromatic properties of their benzene rings. As observed for sulphonylureas, interaction of N-acylphenylalanines with the sulphonylurea receptor does not induce complete inhibition of K_ATP-channel activity in the absence of inhibitory cytosolic nucleotides.

     

The Discovery of MK-4256, a Potent SSTR3 Antagonist as a Potential Treatment of Type 2 Diabetes

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Mechanism of Curcuma longa and Its Neuroactive Components for the Management of Epileptic Seizures: A Systematic Review

Curcuma longa (Turmeric) is a tropical herbaceous perennial plant of the family Zingiberaceae and contains curcuminoids, sesquiterpenoids and monoterpenoids as its major components. Given the broad range of activities that Curcuma longa possesses and also its use as a traditional epilepsy remedy, this review attempts to systematically review the experimentally proven activities of Curcuma longa and its bioactive components, which are related to the management of epileptic seizures. Using the PRISMA model, five databases (Google Scholar, PubMed, ScienceDirect, SCOPUS and SpringerLink) were searched using the keywords [“Curcuma longa” AND “Epilepsy”] and [“Curcuma longa” AND “Seizures”], leaving 34 articles that met the inclusion criteria. The present systematic review elaborated on the experimentally proven potential of Curcuma longa components, such as an aqueous extract of Curcuma longa itself, Curcuma longa oil and active constituents like curcuminoids and bisabolene sesquiterpenoids found in Curcuma longa with anti-seizure potential. Using human equivalent dose calculations, human treatment parameters were suggested for each component by analysing the various studies in this review. This review also determined that the principal components possibly exert their anti-seizure effect via the reduction of corticosterone, modulation of neurotransmitters signalling, modulation of sodium ion channels, reduction of oxidative DNA damage, reduction of lipid peroxidation, upgregulation of brain-derived neurotrophic factor (BDNF) and γ-aminobutyric acid (GABA) mediated inhibition. It is anticipated that this review will help pave the way for future research into the development of Curcuma longa and its neuroactive constituents as potential drug candidates for the management of epilepsy.     

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABAA receptors but with the opposite functionality

1. Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are benzodiazepine site ligands with a second low affinity binding site on GABA_A receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence.
2. In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU-100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU-100079 with a trifluoroethyl substituent produced a positive allosteric response.
3. These functional characteristics were monitored with Cl⁻ currents measurements in cloned rat αxβ2γ2 subtypes of GABA_A receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABA_A receptors with binding of [³⁵S]-TBPS, which is a high affinity ligand specific for GABA_A receptors with exquisite sensitivity to allosteric modulations.
4. This structure-functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

     

Production of β-Cyclocitral and Its Precursor β-Carotene in Microcystis aeruginosa: Variation at Population and Single-Cell Levels

Bloom-forming cyanobacteria produce and release odorous compounds and pose threats to the biodiversity of aquatic ecosystem and to the drinking water supply. In this study, the concentrations of β-cyclocitral in different bacterial growth phases were investigated using GC–MS to determine the growth stage of Microcystis aeruginosa at high risk for β-cyclocitral production. Moreover, the synchronicity of the production of β-cyclocitral and its precursor β-carotene at both population and single-cell levels was assessed. The results indicated that β-cyclocitral was the main odorous compound produced by M. aeruginosa cells. The intracellular concentration of β-cyclocitral (C_β-cc) as well as its cellular quota (Q_β-cc) increased synchronously in the log phase, along with the increase of cell density. However, they reached the maximum values of 415 μg/L and 10.7 fg/cell in the late stationary phase and early stationary phase, respectively. The early stage of the stationary phase is more important for β-cyclocitral monitoring, and the sharp increase in Q_β-cc is valuable for anticipating the subsequent increase in C_β-cc. The molar concentrations of β-cyclocitral and β-carotene showed a linear relationship, with an R² value of 0.92, suggesting that the production of β-cyclocitral was linearly dependent on that of β-carotene, especially during the log phase. However, the increase in Q_β-cc was slower than that in β-carotene during the stationary phase, suggesting that β-cyclocitral production turned to be carotene oxygenase-limited when the growth rate decreased. These results demonstrate that variations of β-cyclocitral production on a single-cell level during different bacterial growth phases should be given serious consideration when monitoring and controlling the production of odorous compounds by M. aeruginosa blooms.     

Pharmacological properties of P2X3-receptors present in neurones of the rat dorsal root ganglia

1. The electrophysiological actions of several agonists which may differentiate between P2X₁- and P2X₃-receptors were studied under concentration and voltage-clamp conditions in dissociated neurones of 1–4 day old rat dorsal root ganglia.
2. β,γ-Methylene-D-ATP (β,γ-me-D-ATP) (1–300 μM), diadenosine 5′,5′′′-P¹,P⁵-pentaphosphate (AP5A) (100 nM–300 μM), diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate (AP4A) (300 nM–300 μM) and uridine 5′-triphosphate (UTP) (1 μM–1 mM) all activated concentration-dependent inward currents with a latency to onset of a few ms.
3. The concentration-response curves for β,γ-me-D-ATP and AP5A and ATP had similar maximum values, while that for AP4A had a lower maximum. The concentration-response curve to UTP was shallow and did not reach a maximum. β,γ-Methylene-L-ATP was virtually inactive. The rank order of agonist potency was ATP>AP5A≈amp;AP4A>β,γ-me-D-ATP>UTP>>β,γ-methylene-L-ATP.
4. The inward currents were inhibited by the P2-receptor antagonists suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (10 μM). PPADS also inhibited responses to ATP (800 nM) and α,β-methylene ATP (2 μM) in a concentration-dependent manner.
5. This study shows that β,γ-me-D-ATP, AP5A, AP4A and UTP all act via a suramin- and PPADS-sensitive P2X-receptor to evoke rapid, transient inward currents in dissociated neurones of rat dorsal root ganglia. The very low activity of β,γ-methylene-L-ATP suggests that the agonists were acting at the P2X₃-subtype to produce these effects.

     

Blockade of peripheral α2-adrenoceptors by L-659,066 enhances glucose tolerance and insulin release in mice

The purpose of this study was to compare the effects of a peripherally selective (L-659,066) vs. a centrally and peripherally active (L-657,743; MK-912) α₂-adrenergic antagonist on murine plasma glucose and insulin levels following fasting or administration of hyperglycemic agents (α₂-adrenergic agonists, glucose). The intravenous administration of L-657,743 or L-659,066 alone did not cause major alterations in plasma glucose or insulin levels. Pretreatment of mice with either of these agents, however, at selective α₂-adrenoceptor blocking doses (0.3–3 mg/kg) prevented the elevations of plasma glucose levels induced by the α₂-adrenoceptor agonists clonidine or 3,4-dihydroxyphenylimino-2-imidazoline (DPl). Prazosin, an α₁-adrenergic antagonist, did not alter clonidine-induced elevations of plasma glucose levels. Pretreatment of fasted mice with L-657,743 or L-659,066 (1 mg/kg) 5 min before receiving intravenous glucose resulted in higher plasma insulin levels and improved glucose tolerance compared to saline-pretreated animals. Moreover, both α₂-adrenergic antagonists enhanced the acute insulin response to glucose. These findings indicate that, under conditions of hyperglycemia, insulin release is enhanced by the blockade of peripheral α₂-adrenoceptors α₂-Adrenoceptor antagonists, such as L-657,743 or the peripherally selective agent L-659,066, may prove effective in treating noninsulin-dependent diabetes mellitus.     

DGAT1 inhibitors protect pancreatic β-cells from palmitic acid-induced apoptosis

Previous studies demonstrated that prolonged exposure to elevated levels of free fatty acids (FFA), especially saturated fatty acids, could lead to pancreatic β-cell apoptosis, which plays an important role in the progression of type 2 diabetes (T2D). Diacylglycerol acyltransferase 1 (DGAT1), an enzyme that catalyzes the final step of triglyceride (TG) synthesis, has been reported as a novel target for the treatment of multiple metabolic diseases. In this study we evaluated the potential beneficial effects of DGAT1 inhibitors on pancreatic β-cells, and further verified their antidiabetic effects in db/db mice. We showed that DGAT1 inhibitors (4a and LCQ908) at the concentration of 1 μM significantly ameliorated palmitic acid (PA)-induced apoptosis in MIN6 pancreatic β-cells and primary cultured mouse islets; oral administration of a DGAT1 inhibitor (4a) (100 mg/kg) for 4 weeks significantly reduced the apoptosis of pancreatic islets in db/db mice. Meanwhile, 4a administration significantly decreased fasting blood glucose and TG levels, and improved glucose tolerance and insulin tolerance in db/db mice. Furthermore, we revealed that pretreatment with 4a (1 μM) significantly alleviated PA-induced intracellular lipid accumulation, endoplasmic reticulum (ER) stress, and proinflammatory responses in MIN6 cells, which might contribute to the protective effects of DGAT1 inhibitors on pancreatic β-cells. These findings provided a better understanding of the antidiabetic effects of DGAT1 inhibitors.