Intra-islet glucagon signalling regulates beta-cell connectivity, first-phase insulin secretion and glucose homoeostasis.

OBJECTIVE: Type 2 diabetes (T2D) is characterised by the loss of first-phase insulin secretion. We studied mice with ß-cell selective loss of the glucagon receptor (Gcgrfl/fl X Ins-1Cre), to investigate the role of intra-islet glucagon receptor (GCGR) signalling on pan-islet [Ca2+]I activity and insulin secretion. METHODS: Metabolic profiling was conducted on Gcgrß-cell-/- and littermate controls. Crossing with GCaMP6f (STOP flox) animals further allowed for ß-cell specific expression of a fluorescent calcium indicator. These islets were functionally imaged in vitro and in vivo. Wild-type mice were transplanted with islets expressing GCaMP6f in ß-cells into the anterior eye chamber and placed on a high fat diet. Part of the cohort received a glucagon analogue (GCG-analogue) for 40 days and the control group were fed to achieve weight matching. Calcium imaging was performed regularly during the development of hyperglycaemia and in response to GCG-analogue treatment. RESULTS: Gcgrß-cell-/- mice exhibited higher glucose levels following intraperitoneal glucose challenge (control 12.7 mmol/L ± 0.6 vs. Gcgrß-cell-/- 15.4 mmol/L ± 0.0 at 15 min, p = 0.002); fasting glycaemia was not different to controls. In vitro, Gcgrß-cell-/- islets showed profound loss of pan-islet [Ca2+]I waves in response to glucose which was only partially rescued in vivo. Diet induced obesity and hyperglycaemia also resulted in a loss of co-ordinated [Ca2+]I waves in transplanted islets. This was reversed with GCG-analogue treatment, independently of weight-loss (n = 8). CONCLUSION: These data provide novel evidence for the role of intra-islet GCGR signalling in sustaining synchronised [Ca2+]I waves and support a possible therapeutic role for glucagonergic agents to restore the insulin secretory capacity lost in T2D.

Glucagon-like peptide 1 (GLP-1).

BACKGROUND: The glucagon-like peptide-1 (GLP-1) is a multifaceted hormone with broad pharmacological potential. Among the numerous metabolic effects of GLP-1 are the glucose-dependent stimulation of insulin secretion, decrease of gastric emptying, inhibition of food intake, increase of natriuresis and diuresis, and modulation of rodent ß-cell proliferation. GLP-1 also has cardio- and neuroprotective effects, decreases inflammation and apoptosis, and has implications for learning and memory, reward behavior, and palatability. Biochemically modified for enhanced potency and sustained action, GLP-1 receptor agonists are successfully in clinical use for the treatment of type-2 diabetes, and several GLP-1-based pharmacotherapies are in clinical evaluation for the treatment of obesity. SCOPE OF REVIEW: In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases. MAJOR CONCLUSIONS: Since its discovery, GLP-1 has emerged as a pleiotropic hormone with a myriad of metabolic functions that go well beyond its classical identification as an incretin hormone. The numerous beneficial effects of GLP-1 render this hormone an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, and neurodegenerative disorders.

Regulation of glucose kinetics during exercise by the glucagon-like peptide-1 receptor.

In response to oral glucose, glucagon-like peptide-1 receptor (Glp1r) knockout (Glp1r−/−) mice become hyperglycaemic due to impaired insulin secretion. Exercise also induces hyperglycaemia in Glp1r−/− mice. In contrast to oral glucose, exercise decreases insulin secretion. This implies that exercise-induced hyperglycaemia in Glp1r−/− mice results from the loss of a non-insulinotropic effect mediated by the Glp1r. Muscle glucose uptake (MGU) is normal in exercising Glp1r−/− mice. Thus, we hypothesize that exercise-induced hyperglycaemia in Glp1r−/− mice is due to excessive hepatic glucose production (HGP). Wild-type (Glp1r+/+) and Glp1r−/− mice implanted with venous and arterial catheters underwent treadmill exercise or remained sedentary for 30 min. [3-3H]glucose was used to estimate rates of glucose appearance (Ra), an index of HGP, and disappearance (Rd). 2[14C]deoxyglucose was used to assess MGU. Glp1r−/− mice displayed exercise-induced hyperglycaemia due to an excessive increase in Ra but normal Rd and MGU. Exercise-induced glucagon levels were ∼2-fold higher in Glp1r−/− mice, resulting in a ∼2-fold higher glucagon:insulin ratio. Since inhibition of the central Glp1r stimulates HGP, we tested whether intracerebroventricular (ICV) infusion of the Glp1r antagonist exendin(9­39) (Ex9) in Glp1r+/+ mice would result in exercise-induced hyperglycaemia. ICV Ex9 did not enhance glucose levels or HGP during exercise, suggesting that glucoregulatory effects of Glp1 during exercise are mediated via the pancreatic Glp1r. In conclusion, functional disruption of the Glp1r results in exercise-induced hyperglycaemia associated with an excessive increase in glucagon secretion and HGP. These results suggest an essential role for basal Glp1r signalling in the suppression of alpha cell secretion during exercise.

Glucagon-like peptide-1 (GLP-1) receptor agonists, obesity and psoriasis: diabetes meets dermatology.

Type 2 diabetes mellitus is characterised by beta cell failure, which frequently develops in the setting of insulin resistance. Inflammation contributes to the pathophysiology of type 2 diabetes by impairing insulin action in peripheral tissues and via reduction of beta cell function. Inflammation may also play an important role in the development of complications that arise in patients with type 2 diabetes. Hence, the anti-inflammatory actions of commonly used glucose-lowering drugs may contribute, indirectly, to their mechanisms of action and therapeutic benefit. Herein we highlight the anti-inflammatory actions of glucagon-like peptide-1 (GLP-1), which exerts direct and indirect actions on immune function. The observations that GLP-1 receptor agonists exert anti-inflammatory actions in preclinical studies, taken together with case reports linking improvements in psoriasis with GLP-1 receptor agonist therapy, illustrates the emerging clinical implications of non-classical anti-inflammatory actions of incretin-based therapeutics.

Metformin regulates the incretin receptor axis via a pathway dependent on peroxisome proliferator-activated receptor-α in mice.

AIMS/HYPOTHESIS: Metformin is widely used for the treatment of type 2 diabetes. Although it reduces hepatic glucose production, clinical studies show that metformin may reduce plasma dipeptidyl peptidase-4 activity and increase circulating levels of glucagon-like peptide 1 (GLP-1). We examined whether metformin exerts glucoregulatory actions via modulation of the incretin axis. METHODS: Metformin action was assessed in Glp1r(-/-), Gipr(-/-), Glp1r:Gipr(-/-), Pparα (also known as Ppara)(-/-) and hyperglycaemic obese wild-type mice with or without the GLP-1 receptor (GLP1R) antagonist exendin(9-39). Experimental endpoints included glucose tolerance, plasma insulin levels, gastric emptying and food intake. Incretin receptor expression was assessed in isolated islets from metformin-treated wild-type and Pparα(-/-) mice, and in INS-1 832/3 beta cells with or without peroxisome proliferator-activated receptor (PPAR)-α or AMP-activated protein kinase (AMPK) antagonists. RESULTS: In wild-type mice, metformin acutely increased plasma levels of GLP-1, but not those of gastric inhibitory polypeptide or peptide YY; it also improved oral glucose tolerance and reduced gastric emptying. Metformin significantly improved oral glucose tolerance despite loss of incretin action in Glp1r(-/-), Gipr(-/-) and Glp1r(-/-) :Gipr(-/-) mice, and in wild-type mice fed a high-fat diet and treated with exendin(9-39). Levels of mRNA transcripts for Glp1r, Gipr and Pparα were significantly increased in islets from metformin-treated mice. Metformin directly increased Glp1r expression in INS-1 beta cells via a PPAR-α-dependent, AMPK-independent mechanism. Metformin failed to induce incretin receptor gene expression in islets from Pparα(-/-) mice. CONCLUSIONS/INTERPRETATION: As metformin modulates multiple components of the incretin axis, and enhances expression of the Glp1r and related insulinotropic islet receptors through a mechanism requiring PPAR-α, metformin may be mechanistically well suited for combination with incretin-based therapies.

Glucagon-like peptide-1 receptor signalling selectively regulates murine lymphocyte proliferation and maintenance of peripheral regulatory T cells.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 receptor (GLP-1R) agonists improve glucose control in animals and humans with type 1 diabetes. However, there is little information on the role of the GLP-1R in the immune system. We studied the role of the GLP-1R in immune function in wild-type (WT) and nonobese diabetic (NOD) and Glp1r-/- mice. METHODS: Glp1r mRNA expression was examined in sorted immune subpopulations by RT-PCR. The effects of GLP-1R activation were assessed on cAMP production and proliferation, migration and survival of primary immune cells from WT and NOD mice. The ability of primary cells from Glp1r-/- mice to proliferate, migrate or survive apoptosis was determined. Immunophenotyping studies were performed to assess the frequency of immune subpopulations in Glp1r-/- mice. RESULTS: Ex vivo activation of the GLP-1R resulted in a modest but significant elevation of cAMP in primary thymocytes and splenocytes from both WT and NOD mice. GLP-1R activation did not increase proliferation of primary thymocytes, splenocytes or peripheral lymph node cells. In contrast, Glp1r-/- thymocytes exhibited a hypoproliferative response, whilst peripheral Glp1r-/- lymphocytes were hyperproliferative in response to mitogenic stimulation. Activation or loss of GLP-1R signalling did not modify apoptosis or chemotaxis in primary lymphocytes. Male Glp1r-/- mice exhibited a significantly lower percentage of peripheral regulatory T cells, although no differences were observed in the numbers of CD4+ and CD8+ T cells and B cells in the spleen and lymph nodes of Glp1r-/- mice. CONCLUSIONS/INTERPRETATION: These studies establish that GLP-1R signalling may regulate lymphocyte proliferation and maintenance of peripheral regulatory T cells.

The glucagon-like peptide 1 receptor is essential for postprandial lipoprotein synthesis and secretion in hamsters and mice.

AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors attenuate postprandial lipaemia through mechanisms that remain unclear. As dyslipidaemia is a contributing risk factor for cardiovascular disease in type 2 diabetes, we examined the mechanisms linking pharmacological and physiological regulation of GLP-1 action to control of postprandial lipid metabolism. METHODS: Postprandial lipid synthesis and secretion were assessed in normal and fructose-fed hamsters and in wild-type mice that were treated with or without sitagliptin. Apolipoprotein B-48 (ApoB-48) synthesis and secretion were also examined in primary enterocyte cultures. The importance of exogenous vs endogenous GLP-1R signalling for regulation of intestinal lipoprotein synthesis and secretion was assessed in mice and hamsters treated with the GLP-1R agonist exendin-4, the GLP-1R antagonist exendin(9-39) and in Glp1r (+/+) vs Glp1r (-/-) mice. RESULTS: Sitagliptin decreased fasting plasma triacylglycerol, predominantly in the VLDL fraction, as well as postprandial triacylglycerol-rich lipoprotein (TRL)-triacylglycerol, TRL-cholesterol and TRL-ApoB-48 in hamsters and mice. GLP-1R activation with exendin-4 alone also decreased plasma and TRL-ApoB-48 in hamsters and mice, and reduced secretion of ApoB-48 in hamster enterocyte cultures. Conversely, blockade of endogenous GLP-1R signalling by the antagonist exendin(9-39) or genetic elimination of GLP-1R signalling in Glp1r (-/-) mice enhanced TRL-ApoB-48 secretion in vivo. Co-administration of exendin(9-39) also abolished the hypolipidaemic effect of sitagliptin. CONCLUSIONS/INTERPRETATION: Potentiation of endogenous incretin action via DPP-4 inhibition or pharmacological augmentation of GLP-1R signalling reduces intestinal secretion of triacylglycerol, cholesterol and ApoB-48. Moreover, endogenous GLP-1R signalling is essential for the control of intestinal lipoprotein biosynthesis and secretion.

Interleukin-1 receptor antagonist gene polymorphism and gingivitis in children.

AIM: To investigate the role of the polymorphism of a variable numbers of tandem repeats of interleukin-1 receptor antagonist gene (IL-1RN) on gingivitis in children. MATERIALS AND METHODS: A total of 146 Caucasian subjects (98 subjects with gingivitis and 48 controls) aged 8-12 years, were enrolled. Plaque and Calculus Indices were recorded to assess the oral hygiene. Gingival and Bleeding on Probing Indices were used to identify patients with gingivitis. DNA was extracted from epithelial cells of the cheek. Normal polymerase chain reaction was used for IL-1Ra genotyping. RESULTS: A significant association was observed between IL-1Ra gene polymorphism and gingivitis in children (P = 0.008). The IL-1RN*2 allele (A2) was significantly more frequent in controls (37%vs 22% in children with gingivitis). In addition, the carriage of A2 seemed to be protective against gingivitis, and it was more frequent in controls (60%vs 40% in children with gingivitis, P = 0.008). Moreover, multiple logistic regression analysis showed that the association between IL-1Ra gene polymorphism and gingivitis in children remained significant (P = 0.014) regardless of the significant influence of plaque (P = 0.013). CONCLUSION: IL-1Ra gene polymorphisms could have an active role in the pathogenesis of gingivitis in Caucasian children and IL-1RN*2 allele could be a protective marker against gingivitis.

Glucagon and glucagon-like peptide receptors as drug targets.

Glucagon and the glucagon-like peptides are derived from a common proglucagon precursor, and regulate energy homeostasis through interaction with a family of distinct G protein coupled receptors. Three proglucagon-derived peptides, glucagon, GLP-1, and GLP-2, play important roles in energy intake, absorption, and disposal, as elucidated through studies utilizing peptide antagonists and receptor knockout mice. The essential role of glucagon in the control of hepatic glucose production, taken together with data from studies employing glucagon antagonists, glucagon receptor antisense oligonucleotides, and glucagon receptor knockout mice, suggest that reducing glucagon action may be a useful strategy for the treatment of type 2 diabetes. GLP-1 secreted from gut endocrine cells controls glucose homeostasis through glucose-dependent enhancement of beta-cell function and reduction of glucagon secretion and gastric emptying. GLP-1 administration is also associated with reduction of food intake, prevention of weight gain, and expansion of beta-cell mass through stimulation of beta-cell proliferation, and prevention of apoptosis. GLP-1R agonists, as well as enzyme inhibitors that prevent GLP-1 degradation, are in late stage clinical trials for the treatment of type 2 diabetes. Exenatide (Exendin-4) has been approved for the treatment of type 2 diabetes in the United States in April 2005. GLP-2 promotes energy absorption, inhibits gastric acid secretion and gut motility, and preserves mucosal epithelial integrity through enhancement of crypt cell proliferation and reduction of epithelial apoptosis. A GLP-2R agonist is being evaluated in clinical trials for the treatment of inflammatory bowel disease and short bowel syndrome. Taken together, the separate receptors for glucagon, GLP-1, and GLP-2 represent important targets for developing novel therapeutic agents for the treatment of disorders of energy homeostasis.

Phenotypic and genotypic identification of enterococci isolated from canals of root-filled teeth with periapical lesions.

The objectives of the present study were to identify enterococcal species isolated from the canals of root-filled teeth with periapical lesions using biochemical and molecular techniques, and to investigate the genetic diversity of the isolates. Twenty-two Enterococcus strains, isolated from the canals of root-filled teeth with persisting periapical lesions, were identified to species level using rapid ID 32 STREP galleries and partial 16S rDNA sequencing. To subtype the strains, genomic DNA from the isolates was analyzed by pulsed field gel electrophoresis (PFGE) after digestion with SmaI. Intragenic regions of two genes, ace and salA, were sequenced for further differentiation of the isolates. All strains were identified as Enterococcus faecalis by both commercial kit and partial 16S rDNA sequencing. PFGE with SmaI of 22 isolates demonstrated 18 macrorestriction profiles, whereas 13 distinct genotypes were identified after analysis of the ace and salA composite sequences. Most of the isolates from distinct patients had different PFGE profiles. Moreover, in two cases, different E. faecalis strains were found in different root-filled teeth from the same mouth. E. faecalis was the only enterococcal species isolated from the canals of root-filled teeth with periapical lesions. Genetic heterogeneity was observed among the E. faecalis isolates following PFGE and sequence-based typing method. Furthermore, the genetic diversity within root canal strains was similar to previous reports regarding E. faecalis isolates from different clinical and geographic origins.

Molecular species of phosphatidylethanolamine from continuous cultures of Saccharomyces pastorianus syn. carlsbergensis strains.

Saccharomyces pastorianus syn. carlsbergensis strain 34/70 is well known to be the most used strain for lager beer production. The difference between this strain and very closely related strain 34/78 is the latter's greater flocculating character. This single physiological trait can cause technical difficulties in beer production. The aim of this study was to determine whether lipid analysis by a combination of thin layer chromatography (TLC) with electrospray ionization mass spectrometry (ESI-MS) could be used as a strain-typing technique in order to distinguish S. pastorianus syn. carlsbergensis strain 34/70 from strain 34/78. Both strains (34/70 and 34/78) were harvested after continuous culture under standard conditions. Polar lipids were then extracted from lyophilized cultures and analysed by TLC in order to separate phospholipid families. Phosphatidylethanolamine (PE) was extracted and investigated using ESI-MS, to gain further information on individual molecular species. Using TLC analysis, lipids were separated corresponding to standards for PE, phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA) and sphingomyelin (SM). ESI-MS of the PE band, separated by TLC, showed that electrospray mass spectra were highly reproducible for repeat cultures. Novel findings were that both brewing strains displayed major phospholipid peaks with m/z 714, PE (34 : 2) m/z 742, PE (36 : 2) and m/z 758, PE (37 : 1). However, strain 34/78 had additional peaks of m/z 700, PE (33 : 2) and m/z 728, PE (35 : 2). Strain 34/70 had an extra peak with m/z 686 PE (32 : 2). We conclude that combined TLC/ESI-MS can distinguish between S. pastorianus syn. carlsbergensis 34/70 and 34/78 and may be a useful typing technique for differentiation of closely related yeast strains. This novel approach may aid quality assurance and could be suitable for yeast collections and larger industrial companies.

Comparison of polar lipids from yeast and mycelial forms of Candida albicans and Candida dubliniensis.

The aim of the present study was to compare polar lipids of yeast and mycelial forms of both Candida albicans and Candida dubliniensis. Cultures were harvested from Lee's medium after incubation at 37 degrees C for 48 h. Yeast and mycelial forms were washed, separated from one another, dried and lipids extracted and prepared for fast atom bombardment mass spectrometry analysis in the negative-ion mode. For fatty acids, differences between the yeast and mycelial forms were greater for C. dubliniensis than for C. albicans. For the phospholipid families, phosphatidic acid and phosphatidylethanolamine, differences between yeast and mycelial forms were greater for C. dubliniensis than for C. albicans. For both species, it is concluded that both fatty acid and phospholipid molecular species compositions differ according to whether the cells are in the yeast or mycelial form.

The relationship between interleukin-10 gene polymorphism at position -1082 and susceptibility to gingivitis in children.

BACKGROUND: Interleukin (IL)-10 is an anti-inflammatory cytokine. The protective role of this cytokine against different diseases has been demonstrated in several studies. However, no such study has been carried out on gingivitis. The objective of this study was to determine whether differences exist between Caucasian children with and without gingivitis in the distribution of IL-10 alleles at position -1082. METHODS: A total of 260 Caucasian children (86 controls, 174 patients), aged 8 to 12 years, from the University Dental Hospital of Manchester, U.K., were examined. Plaque (PI), calculus (CI), gingival (GI), and bleeding on probing (BOP) indices were used to assess gingival health. DNA was obtained from buccal epithelial cells. Amplification refractory mutation system polymerase chain reaction (ARMS-PCR) was used for genotyping IL-10 polymorphism. Chi square tests were carried out to test the association between allele and genotype frequencies and the severity of gingivitis. Multiple logistic regression was used to determine the role of IL-10 gene polymorphism at position -1082 while adjusting for potential confounders such as plaque, age, and gender. RESULTS: Gingivitis was present in 67% of the children examined. Frequencies of alleles -1082*A and -1082*G were 45% and 55%, respectively. An increased risk of having gingivitis was found in allele A positive children (G/A, A/A); 75% versus 25% in allele A negative children (G/G); (P = 0.01). The -1082*A allele was significantly more common in children with gingivitis; 49% versus 37% in controls (P = 0.01). Multivariate logistic regression analysis showed that allele A remained a risk factor for gingivitis in children (P = 0.03) regardless of plaque or age. Also, allele A positive children were at increased odds of having gingivitis of 1.8 (95% confidence interval [CI]: 1.05 to 3.06) compared to allele A negative children after adjusting for plaque, age, and gender. CONCLUSION: These data suggest that the -1082*A allele could be a risk factor for gingivitis.

The HeLa cell glucagon-like peptide-2 receptor is coupled to regulation of apoptosis and ERK1/2 activation through divergent signaling pathways.

Glucagon-like peptide-2 (GLP-2) regulates proliferative and cytoprotective pathways in the intestine; however GLP-2 receptor (GLP-2R) signal transduction remains poorly understood, and cell lines that express the endogenous GLP-2R have not yet been isolated. We have now identified several expressed sequence tags from human cervical carcinoma cDNA libraries that correspond to GLP-2R nucleotide sequences. GLP-2R mRNA transcripts were detected by RT-PCR in two human cervical carcinoma cell lines, including HeLa cells. GLP-2 increased cAMP accumulation and activated ERK1/2 in HeLa cells transiently expressing the cloned human HeLa cell GLP-2R cDNA. However, the GLP-2R-induced activation of ERK1/2 was not mediated through Galphas, adenylyl cyclase, or transactivation of the epidermal growth factor receptor, but was pertussis toxin sensitive, inhibited by dominant negative Ras, and dependent on betagamma-subunits. GLP-2 also induced a significant increase in bromodeoxyuridine incorporation that was blocked by dominant negative Ras. Furthermore, GLP-2 inhibited HeLa cell apoptosis induced by LY294002 in a protein kinase A-dependent, but ERK-independent, manner. These findings demonstrate that the HeLa cell GLP-2R differentially signals through both Galphas/cAMP- and Gi/Go-dependent pathways, illustrating for the first time that the GLP-2R is capable of coupling to multiple heterotrimeric G proteins defining distinct GLP-2R-dependent biological actions.

Characterization and functional role of voltage gated cation conductances in the glucagon-like peptide-1 secreting GLUTag cell line.

Glucagon-like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion. It is currently under therapeutic evaluation because it enhances insulin secretion in type 2 diabetes. Previous studies using the GLP-1 secreting cell line GLUTag have shown that the cells are electrically active, and that the action potential frequency is regulated by nutrients. In this study we characterize voltage gated currents underlying this electrical activity and correlate the electrophysiological findings with gene expression determined by microarrays. Whole cell voltage clamp experiments designed to separate different ionic components revealed rapidly inactivating sodium currents sensitive to tetrodotoxin, calcium currents sensitive to nifedipine and omega-conotoxin GVIA, and sustained as well as rapidly inactivating potassium currents, which were sensitive to TEA and 4-AP, respectively. In perforated patch experiments we also observed hyperpolarization-activated currents which were inhibited by ZD7288. The amplitude of the sodium current was approximately 10 times that of the other depolarizing currents and tetrodotoxin abolished action potential firing. In secretion experiments, however, nifedipine, but not tetrodotoxin, omega-conotoxin GVIA or ZD7288, inhibited glucose-induced GLP-1 release. Consistent with this finding, the intracellular Ca2+ response to glucose was impaired by nifedipine but not by tetrodotoxin. Thus, in GLUTag cells, GLP-1 release is not dependent on the firing of Na+-carrying action potentials but requires membrane depolarization and Ca2+ entry through L-type Ca2+ channels. Understanding the characteristics of the currents and the molecular identification of the underlying channels in GLP-1 secreting cells might facilitate the development of agents to enhance GLP-1 secretion in vivo.

Antimicrobial susceptibility of Enterococcus faecalis isolated from canals of root filled teeth with periapical lesions.

AIM: To test, in vitro, the susceptibility to different antibiotics of Enterococcus faecalis isolates from canals of root filled teeth with periapical lesions. METHODOLOGY: Twenty-one E. faecalis isolates, from canals of root filled teeth with persisting periapical lesions, were tested for their antibiotic susceptibilities. The following antibiotics were used: benzylpenicillin, amoxicillin, amoxicillin-clavulanic acid, erythromycin, azithromycin, vancomycin, chloramphenicol, tetracycline, doxycycline, ciprofloxacin and moxifloxacin. Minimal inhibitory concentrations (MICs) for the antimicrobial agents were determined using the E-test System (AB BIODISK, Solna, Sweden), and the E. faecalis strains classified as susceptible or resistant according to the guidelines of National Committee for Clinical Laboratory Standards (NCCLS). The strains were also tested for beta-lactamase production with nitrocefin (Oxoid, Basingstoke, UK). RESULTS: All strains were susceptible to penicillins in vitro, however, the MICs of amoxicillin and amoxicillin-clavulanic acid (MIC(90) = 0.75 microg mL(-1)) were lower than for benzylpenicillin (MIC(90) = 3.0 microg mL(-1)). All strains studied were also susceptible to vancomycin and moxifloxacin, whilst 95.2% were susceptible to chloramphenicol. Amongst the isolates, 85.7% were susceptible to tetracycline and doxycycline and 80.9% to ciprofloxacin. The MIC of erythromycin ranged from 0.38 to >256 microg mL(-1); only 28.5% of the strains were susceptible (MIC < or = 0.5 microg mL(-1)). Limited susceptibility was also observed with azithromycin which was active against only 14.2% of isolates. No strains produced beta-lactamase. CONCLUSION: Enterococcus faecalis isolates were completely susceptible, in vitro, to amoxicillin, amoxicillin-clavulanic acid, vancomycin and moxifloxacin. Most isolates were susceptible to chloramphenicol, tetracycline, doxycycline or ciprofloxacin. Erythromycin and azithromycin were least effective.

Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A. naeslundii, Candida albicans and Enterococcus faecalis.

AIM: To determine the resistance of microorganisms associated with refractory endodontic infections to sodium hypochlorite used as a root canal irrigant. METHODOLOGY: Two strains each of Actinomyces naeslundii, Candida albicans and Enterococcus faecalis were tested as late logarithmic phase inocula, against sodium hypochlorite adjusted to 0.5, 1.0, 2.5 and 5.25% w/v. Contact times used were 0, 10, 20, 30, 60 and 120 s. In the case of E. faecalis, additional experiments used contact times of 1.0, 2.0, 5.0, 10.0 and 30.0 min. Anti-microbial action was halted by sodium thiosulphate addition. Survivors were measured primarily using viable counts on drop plates. Additionally, pour plates were used to count low colony-forming units (cfu) and dilutions to 10(-6) were used to count high cfu. RESULTS: All concentrations of NaOCl lowered cfu below the limit of detection after 10 s in the case of A. naeslundii and C. albicans. However, E. faecalis proved to be more resistant to NaOCl. Using 0.5% NaOCl for 30 min reduced cfu to zero for both strains tested. This compares with 10 min for 1.0%, 5 min for 2.5% and 2 min for 5.25% (P < 0.001). Regression analysis for the dependent variable log(e)(count + 1) with log(e)(time + 1) and concentration as explanatory variables gave rise to a significant interaction between time and concentration (P < 0.001). CONCLUSION: The published association of E. faecalis with refractory endodontic infection may result, at least partially, from high resistance of this species to NaOCl. This does not appear to be the case with A. naeslundii or C. albicans.

Double incretin receptor knockout (DIRKO) mice reveal an essential role for the enteroinsular axis in transducing the glucoregulatory actions of DPP-IV inhibitors.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are gut-derived incretins that potentiate glucose clearance following nutrient ingestion. Elimination of incretin receptor action in GIPR(-/-) or GLP-1R(-/-) mice produces only modest impairment in glucose homeostasis, perhaps due to compensatory upregulation of the remaining incretin. We have now studied glucose homeostasis in double incretin receptor knockout (DIRKO) mice. DIRKO mice exhibit normal body weight and fail to exhibit an improved glycemic response after exogenous administration of GIP or the GLP-1R agonist exendin-4. Plasma glucagon and the hypoglycemic response to exogenous insulin were normal in DIRKO mice. Glycemic excursion was abnormally increased and levels of glucose-stimulated insulin secretion were decreased following oral but not intraperitoneal glucose challenge in DIRKO compared with GIPR(-/-) or GLP-1R(-/-) mice. Similarly, glucose-stimulated insulin secretion and the response to forskolin were well preserved in perifused DIRKO islets. Although the dipeptidyl peptidase-IV (DPP-IV) inhibitors valine pyrrolidide (Val-Pyr) and SYR106124 lowered glucose and increased plasma insulin in wild-type and single incretin receptor knockout mice, the glucose-lowering actions of DPP-IV inhibitors were eliminated in DIRKO mice. These findings demonstrate that glucose-stimulated insulin secretion is maintained despite complete absence of both incretin receptors, and they delineate a critical role for incretin receptors as essential downstream targets for the acute glucoregulatory actions of DPP-IV inhibitors.

Extrahypothalamic expression of the glucagon-like peptide-2 receptor is coupled to reduction of glutamate-induced cell death in cultured hippocampal cells.

Proglucagon-derived glucagon-like peptide-2 (GLP-2) is liberated in enteroendocrine cells and neurons. GLP-2 regulates energy absorption and epithelial integrity in the gastrointestinal tract, whereas GLP-2 action in the central nervous system remains poorly defined. We identified proglucagon and GLP-2 receptor (GLP-2R) mRNA transcripts by RT-PCR in multiple regions of the developing and adult rat central nervous system. GLP-2R mRNA transcripts were localized by in situ hybridization to the hippocampus, hypothalamus, nucleus of the solitary tract, parabrachial nucleus, supramammillary nucleus, and substantia nigra. The bioactive form of GLP-2, GLP-2-(1-33) was detected by RIA and HPLC analysis in the fetal and adult brainstem and hypothalamus. GLP-2 stimulated increases in cAMP accumulation in postnatal d 8, but not embryonic d 14, dispersed neonatal rat brainstem tissues. The actions of GLP-2 were independent of the GLP-1R antagonist exendin-(9-39), and GLP-2 stimulated cAMP accumulation in hippocampal cell cultures from both wild-type and GLP-1R(-/-) mice. GLP-2 significantly reduced glutamate-induced excitotoxic injury in hippocampal cells via a protein kinase A-dependent pathway, but had no effect on the rate of cell proliferation. These findings establish the presence of a functional GLP-2-GLP-2R axis in the developing rodent brain and demonstrate that GLP-2 exerts cytoprotective actions in cells derived from the central nervous system.