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 and energy expenditure; Revisiting amino acid metabolism and implications for weight loss therapy.

Glucagon receptor (GCGR)-targeted multi-agonists are being developed for the treatment of obesity and metabolic disease. GCGR activity is utilised for its favourable weight loss and metabolic properties, including increased energy expenditure (EE) and hepatic lipid metabolism. GLP1R and GIPR activities are increasingly present in a multi-agonist strategy. Due to the compound effect of increased satiety, reduced food intake and increased energy expenditure, the striking weight loss effects of these multi-agonists has been demonstrated in pre-clinical models of obesity. The precise contribution and mechanism of GCGR activity to enhanced energy expenditure and weight loss in both rodents and humans is not fully understood. In this review, our understanding of glucagon-mediated EE is explored, and an amino acid-centric paradigm contributing to this phenomenon is presented. The current progress of GCGR-targeted multi-agonists in development is also highlighted with a focus on the implications of glucagon-stimulated hypoaminoacidemia.

Inhibitory effect and mechanism of Lactobacillus crispatus on cervical precancerous cells Ect1/E6E7 and screening of early warning factors.

OBJECTIVE: To study the potential mechanism of Lactobacillus crispatus inhibiting cervical squamous intraepithelial lesion (SIL) and screen the early warning factors of SIL. METHODS: The effects of Lactobacillus crispatus on the proliferation, apoptosis, cross pore migration and invasion and cytokines of cervical precancerous cells Ect1/E6E7 were detected respectively. The effect of Lactobacillus crispatus on the expression of differential proteins screened in Ect1/E6E7 cells were detected by Western blot. RESULTS: Lactobacillus crispatus significantly inhibited the proliferation, induced apoptosis and inhibited cell migration of Ect1/E6E7 cells in a time-dependent manner (P < 0.05), but had no significant effect on cell invasion. Lactobacillus crispatus significantly promoted the secretion of Th1 cytokines and inhibited the secretion of Th2 cytokines by Ect1/E6E7 cells (P < 0.05). In addition, compared with SiHa cells in the control group, the expression of differential proteins PCNA, ATM, LIG1 and HMGB1 in Ect1/E6E7cells decreased significantly, while the expression of TDG and OGG1 proteins increased significantly (P < 0.05). ABCG2 protein in Ect1/E6E7 cells was slightly higher than that in SiHa cells, but the difference was not statistically significant. What is interesting is that Lactobacillus crispatus significantly inhibited the expression of ABCG2, PCNA, ATM, LIG1, OGG1 and HMGB1 proteins in Ect1/E6E7 cells, and promoted the expression of TDG protein. CONCLUSIONS: Lactobacillus crispatus may inhibit the function of Ect1/E6E7 cells through multiple pathways and exert the potential to reverse the progression of SIL.

Glucagon increases energy expenditure independently of brown adipose tissue activation in humans.

AIMS: To investigate, for a given energy expenditure (EE) rise, the differential effects of glucagon infusion and cold exposure on brown adipose tissue (BAT) activation in humans. METHODS: Indirect calorimetry and supraclavicular thermography was performed in 11 healthy male volunteers before and after: cold exposure; glucagon infusion (at 23 °C); and vehicle infusion (at 23 °C). All volunteers underwent (18)F-fluorodeoxyglucose ((18)F-FDG) positron emission tomography (PET)/CT scanning with cold exposure. Subjects with cold-induced BAT activation on (18)F-FDG PET/CT (n = 8) underwent a randomly allocated second (18)F-FDG PET/CT scan (at 23 °C), either with glucagon infusion (n = 4) or vehicle infusion (n = 4). RESULTS: We observed that EE increased by 14% after cold exposure and by 15% after glucagon infusion (50 ng/kg/min; p < 0.05 vs control for both). Cold exposure produced an increase in neck temperature (+0.44 °C; p < 0.001 vs control), but glucagon infusion did not alter neck temperature. In subjects with a cold-induced increase in the metabolic activity of supraclavicular BAT on (18)F-FDG PET/CT, a significant rise in the metabolic activity of BAT after glucagon infusion was not detected. Cold exposure increased sympathetic activation, as measured by circulating norepinephrine levels, but glucagon infusion did not. CONCLUSIONS: Glucagon increases EE by a similar magnitude compared with cold activation, but independently of BAT thermogenesis. This finding is of importance for the development of safe treatments for obesity through upregulation of EE.

Can Bayliss and Starling gut hormones cure a worldwide pandemic?

Bayliss and Starling first coined the term 'hormone' with reference to secretin, a substance they found that was produced by the gut, but released into the blood stream to act at a distance. The intestine is now known as the largest endocrine organ in the body, and it produces numerous hormones with a wide range of functions. These include controlling appetite and energy homeostasis. Obesity is one of the greatest health threats facing the world today. At present, the only successful treatment is surgery. Bariatric procedures such as the Roux-en-Y bypass work by elevating gut hormones that induce satiety. Significant research has gone into producing versions of these hormones that can be delivered therapeutically to treat obesity. This review looks at the role of gut hormones in obesity, and the development of gut hormone-derived obesity treatments.

An expression signature of the angiogenic response in gastrointestinal neuroendocrine tumours: correlation with tumour phenotype and survival outcomes.

BACKGROUND: Gastroenteropancreatic neuroendocrine tumours (GEP-NETs) are heterogeneous with respect to biological behaviour and prognosis. As angiogenesis is a renowned pathogenic hallmark as well as a therapeutic target, we aimed to investigate the prognostic and clinico-pathological role of tissue markers of hypoxia and angiogenesis in GEP-NETs. METHODS: Tissue microarray (TMA) blocks were constructed with 86 tumours diagnosed from 1988 to 2010. Tissue microarray sections were immunostained for hypoxia inducible factor 1α (Hif-1α), vascular endothelial growth factor-A (VEGF-A), carbonic anhydrase IX (Ca-IX) and somatostatin receptors (SSTR) 1-5, Ki-67 and CD31. Biomarker expression was correlated with clinico-pathological variables and tested for survival prediction using Kaplan-Meier and Cox regression methods. RESULTS: Eighty-six consecutive cases were included: 51% male, median age 51 (range 16-82), 68% presenting with a pancreatic primary, 95% well differentiated, 51% metastatic. Higher grading (P=0.03), advanced stage (P<0.001), high Hif-1α and low SSTR-2 expression (P=0.03) predicted for shorter overall survival (OS) on univariate analyses. Stage, SSTR-2 and Hif-1α expression were confirmed as multivariate predictors of OS. Median OS for patients with SSTR-2+/Hif-1α-tumours was not reached after median follow up of 8.8 years, whereas SSTR-2-/Hif-1α+ GEP-NETs had a median survival of only 4.2 years (P=0.006). CONCLUSION: We have identified a coherent expression signature by immunohistochemistry that can be used for patient stratification and to optimise treatment decisions in GEP-NETs independently from stage and grading. Tumours with preserved SSTR-2 and low Hif-1α expression have an indolent phenotype and may be offered less aggressive management and less stringent follow up.

Immunohistochemical markers of the hypoxic response can identify malignancy in phaeochromocytomas and paragangliomas and optimize the detection of tumours with VHL germline mutations.

BACKGROUND: There are no reliable markers of malignancy in phaeochromocytomas (PCC) and paragangliomas (PGL). We investigated the relevance of the mammalian target of rapamycin (mTOR)/AKT and hypoxic pathways as novel immunohistochemical markers of malignancy. METHODS: Tissue microarray blocks were constructed with a total of 100 tumours (10 metastatic) and 20 normal adrenomedullary samples. Sections were immunostained for hypoxia-inducible factor 1α (Hif-1α), vascular endothelial growth factor A (VEGF-A), mTOR, carbonic anhydrase IX (CaIX) and AKT. The predictive performance of these markers was studied using univariate, multivariate and receiver operating characteristic analyses. RESULTS: In all, 100 consecutive patients, 64% PCC, 29% familial with a median tumour size of 4.7 cm (range 1-14) were included. Univariate analyses showed Hif-1α overexpression, tumour necrosis, size >5 cm, capsular and vascular invasion to be predictors of metastasis. In multivariate analysis, Hif-1α, necrosis and vascular invasion remained as independent predictors of metastasis. Hif-1α was the most discriminatory biomarker for the presence of metastatic diffusion. Strong membranous CaIX expression was seen in von Hippel-Lindau (VHL) PCC as opposed to other subtypes. CONCLUSION: Lack of vascular invasion, tumour necrosis and low Hif-1α expression identify tumours with lower risk of malignancy. We propose membranous CaIX expression as a potential marker for VHL disease in patients presenting with PCC.

Genome-wide expression profiling of RNA interference of hepatitis B virus gene expression and replication.

Small interfering RNA (siRNA) has been used repeatedly to down-regulate viral gene expression and inhibit viral replication in mammalian cells. In this study, we showed that siRNAs specific for two conserved regions within the hepatitis B S antigen (HBsAg) gene can inhibit antigen production in two human liver cell lines which constitutively produce and secrete HBsAg. The inhibitory effect was concentration dependent for both PLC/PRF/5 and 2.2.15 cells. Decreases in the corresponding viral transcript levels were observed. The inhibitory effect was observed within 24 h and was still evident 7 days after the initial treatment with siRNA. A significant reduction in virion production was also observed for the 2.2.15 cells. A critical consideration in this study was the specificity of the siRNA-mediated inhibition. To address this, we first examined the effects on cell growth and viability. These were not affected in either cell line. cDNA microarrays were also used to examine genome-wide changes in gene regulation. No significant off-target gene regulation was observed in either cell line. Our findings thus indicate that siRNA can specifically mediate the down-regulation of viral gene expression leading to a reduction in virion production.

BpeAB-OprB, a multidrug efflux pump in Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents, including beta-lactams, aminoglycosides, macrolides, and polymyxins. An operon, bpeR-bpeA-bpeB-oprB, which encodes a putative repressor, a membrane fusion protein, an inner membrane protein, and an outer membrane protein, respectively, of a multidrug efflux pump of the resistance-nodulation-division family was identified in B. pseudomallei. The divergently transcribed bpeR gene encodes a putative repressor protein of the TetR family which probably regulates the expression of the bpeAB-oprB gene cluster. Comparison of the MICs and minimal bactericidal concentrations of antimicrobials for bpeAB deletion mutant KHW Delta bpeAB and its isogenic wild-type parent, KHW, showed that the B. pseudomallei BpeAB-OprB pump is responsible for the efflux of the aminoglycosides gentamicin and streptomycin, the macrolide erythromycin, and the dye acriflavine. Antibiotic efflux by the BpeAB-OprB pump was dependent on a proton gradient and differs from that by the AmrAB-OprA pump in that it did not efflux the aminoglycoside spectinomycin or the macrolide clarithromycin. The broad-spectrum efflux pump inhibitor MC-207,110 did not potentiate the effectiveness of the antimicrobials erythromycin and streptomycin in B. pseudomallei.

Mitochondrial respiratory function and antioxidant capacity in normal and cirrhotic livers following partial hepatectomy.

For many liver malignancies, major hepatectomy is the usual therapy. Although a normal liver has a tremendous capacity for regeneration, liver hepatectomy in humans is usually carried out on a diseased liver and, in such cases, liver regeneration takes place in a cirrhotic remnant. Mitochondrial function in cirrhotic livers shows a variety of changes compared to control livers. This study investigated how mitochondrial respiratory function and antioxidant capacity change following partial hepatectomy of cirrhotic livers, because liver regeneration requires greater energy demands and control of oxidative stress. Cirrhosis was induced in male Wistar-Furth rats by administration of thioacetamide. NADH-cytochrome c reductase activity, mitochondrial glutathione peroxidase activity and mitochondrial GSH levels were all significantly lowered in cirrhotic livers and in the cirrhotic remnants up to 72 h after 70% hepatectomy when compared to the corresponding controls. Lower respiratory control ratios with succinate as substrate were also observed from 6 to 48 h post-hepatectomy. At 24 h post-hepatectomy, higher levels of lipid peroxidation were observed. We conclude that, compared to the controls, cirrhotic livers have diminished oxidative phosphorylation capabilities due to changes in NADH and FADH(2)-linked respiration as well as impaired antioxidant defenses following partial hepatectomy. Both of these factors, if critical, could then impede liver regeneration.

Mechanism of clofibrate hepatotoxicity: mitochondrial damage and oxidative stress in hepatocytes.

Peroxisome proliferators have been found to induce hepatocarcinogenesis in rodents, and may cause mitochondrial damage. Consistent with this, clofibrate increased hepatic mitochondrial oxidative DNA and protein damage in mice. The present investigation aimed to study the mechanism by which this might occur by examining the effect of clofibrate on freshly isolated mouse liver mitochondria and a cultured hepatocyte cell line, AML-12. Mitochondrial membrane potential (Delta Psi(m)) was determined by using the fluorescent dye 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) and tetramethylrhodamine methyl ester (TMRM). Application of clofibrate at concentrations greater than 0.3 mM rapidly collapsed the Delta Psi(m) both in liver cells and in isolated mitochondria. The loss of Delta Psi(m) occurred prior to cell death and appeared to involve the mitochondrial permeability transition (MPT), as revealed by calcein fluorescence studies and the protective effect of cyclosporin A (CsA) on the decrease in Delta Psi(m). Levels of reactive oxygen species (ROS) were measured with the fluorescent probes 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (DCFDA) and dihydrorhodamine 123 (DHR123). Treatment of the hepatocytes with clofibrate caused a significant increase in intracellular and mitochondrial ROS. Antioxidants such as vitamin C, deferoxamine, and catalase were able to protect the cells against the clofibrate-induced loss of viability, as was CsA, but to a lesser extent. These results suggest that one action of clofibrate might be to impair mitochondrial function, so stimulating formation of ROS, which eventually contribute to cell death.

The efficiency of absorbable clips in minimally invasive surgery.

This study was conducted to evaluate the efficiency of polymeric clips in minimally invasive surgery. Absorbable polymeric clips were applied to the cystic duct, appendicular stump, and blood vessels in 35 patients undergoing laparoscopic or thoracoscopic procedures. All patients were reviewed 3 months after their operations, and no complications related to the clips were observed. These findings indicate that absorbable clips have more advantages than metallic clips. We believe that as this clip is easy to operate with safety, it should be used in the majority of minimally invasive surgical procedures.

Detection and species determination of malaria parasites by PCR: comparison with microscopy and with ParaSight-F and ICT malaria Pf tests in a clinical environment.

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients. Plasmodium infections were identified with a genus-specific primer set, and species differentiation between Plasmodium falciparum and Plasmodium vivax was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed P. falciparum-P. vivax infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays for P. falciparum, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive for P. vivax at the time of discharge were later readmitted to the hospital with a recurrence of P. vivax infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria.

Biologic activity of oligonucleotides with polarity and anomeric center reversal.

Human papillomavirus (HPV) type 16 E6 and E7 inactivate the tumor suppressors p53 and pRB, respectively. Both viral oncoproteins play important roles in maintaining the transformed phenotype of cells. In this study, we examine the effects of antisense oligodeoxynucleotides with polarity and anomeric center reversal (alpha/beta-ODNs). ODNs of the general structure 5'alphaN3'3'NNN5'5'alphaN3'3'NNNN5'5'alphaN3+ ++'3'N5' were synthesized using phosphoramidite DNA chemistry. These alpha/beta-ODNs were complementary in sequence to regions flanking the start codons of HPV type 16 E6 and E7 genes. The anti-HPV type 16 alpha/beta-ODNs were able to form stable duplexes with their complementary RNA, which then serve as substrates for RNase H hydrolysis. Anti-HPV type 16 alpha/beta-ODNs also specifically inhibited the growth of two cervical carcinoma cell lines, CaSki and SiHa, both of which harbor HPV type 16 DNA. A decrease in E7 protein expression was also observed. Injection of nude mice with SiHa cells induces tumors. Treatment of these tumor-bearing mice with anti-HPV type 16 alpha/beta-ODNs led to substantially smaller tumors. These results show that alpha/beta-ODNs can exert antisense activities both in vitro and in vivo on the E6 and E7 genes of HPV type 16.

Partial nucleotide sequence and organisation of extrachromosomal plastid-like DNA in Plasmodium berghei.

The murine malaria parasite Plasmodium berghei contains a plastid-like extrachromosomal genome. This genome is 30.7 kb in size and is transcriptionally active as shown by RT-PCR. DNA sequence analysis of the genome reveals 69.9-95.5% homology to sequences of the 35-kb extrachromosomal circle found in the human malaria species Plasmodium falciparum. Homologous sequences include regions of genes for the ssu-rRNA, lsu-rRNA, rpo B and clusters of t-RNAs. Sequence variation between the two Plasmodium species exists in the non-coding interspacing regions. A physical map has been constructed for the P. berghei circle, indicating the EcoRI and HindIII restriction sites as well as the arrangement of the rRNA, rpo B and tRNA genes. Arrangement of these genes is similar to that found on the P. falciparum 35-kb circle. The P. berghei circular element is distinct from the mitochondrial 6-kb DNA of both the murine and the human Plasmodium species. Preliminary results indicate that the circle may be a useful target for drug therapy.

Direct PCR amplification and sequence analysis of extrachromosomal Plasmodium DNA from dried blood spots.

The Plasmodium parasite possesses two extrachromosomal genomes; the mitochondrial genetic element and the extrachromosomal plastid-like DNA. The latter has only been fully described for one culture strain of P. falciparum. In this study, a rapid procedure for amplifying plastid DNA from dried blood spots of blood infected with different malaria species was developed. PCR amplification of a 595 bp fragment within the plastid-like large subunit ribosomal-RNA (LSU-rRNA) gene was achieved using primers derived from the P. falciparum sequence. The PCR product was observed in all Plasmodium species examined. Sequence analysis of amplified products homologous to an LSU-rRNA fragment of the plastid-like extrachromosomal circle revealed extensive conservation between Plasmodium species including P. falciparum, P. vivax, P. malariae and P. berghei.

ELISA for the detection of serum and saliva IgA against the BMRFI gene product of Epstein-Barr virus.

The BMRF1 protein is an Epstein-Barr virus (EBV) DNA polymerase accessory protein that forms part of the early antigen diffuse (EA-D) component. An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of IgA antibody to the BMRF1 protein of EBV in saliva and serum samples. The assay was shown to be specific for nasopharyngeal carcinoma (NPC) patients and, when used with saliva alone, to have a sensitivity comparable to an existing indirect immunoperoxidase assay for early antigens. The sensitivity of the assay could be significantly enhanced to 86% by the use of paired saliva and serum samples.

Identification of a Plasmodium berghei antigen sharing common features with P. falciparum and P. chabaudi parasitophorous-vacuole membrane antigens.

On the basis of immunological cross-reactivity, we identified a 43-kDa Plasmodium berghei antigen with homology to the exp-1 antigen from P. falciparium. The P. berghei antigen was recognized by an antibody directed against an epitope on the C-terminus of the P. falciparum exp-1 protein. This antigen is localized on the surface of the parasite and shares peptide sequence homology with the P. chabudi antigen Ag3008. To investigate further the role of the P. berghei antigen, we designed antisense phosphorothioate oligodeoxynucleotides (PS oligos) complementary to sequences of the exp-1 mRNA from P. falciparum. The PS oligos were capable of inhibiting the development of P. falciparum in vitro by 47%. In vivo, experiments in mice showed that the same PS oligos had the potential to extend the life span of mice infected with P. berghei by a factor of 2-4. The immunological cross-reactivity and the antisense inhibition of P. berghei parasite development in vivo indicate that this antigen may be a homologue of exp-1 from P. falciparum that has functional importance for parasite survival.