Development of carbon monoxide-releasing molecules conjugated to polysaccharides (glyco-CORMs) for delivering CO during obesity.

Metal carbonyls have been developed as carbon monoxide-releasing molecules (CO-RMs) to deliver CO for therapeutic purposes. The manganese-based CORM-401 has been recently reported to exert beneficial effects in obese animals by reducing body weight gain, improving glucose metabolism and reprogramming adipose tissue towards a healthy phenotype. Here, we report on the synthesis and characterization of glyco-CORMs, obtained by grafting manganese carbonyls on dextrans (70 and 40 kDa), based on the fact that polysaccharides facilitate the targeting of drugs to adipose tissue. We found that glyco-CORMs efficiently deliver CO to cells in vitro with higher CO accumulation in adipocytes compared to other cell types. Oral administration of two selected glyco-CORMs (5b and 6b) resulted in CO accumulation in various organs, including adipose tissue. In addition, glyco-CORM 6b administered for eight weeks elicited anti-obesity and positive metabolic effects in mice fed a high fat diet. Our study highlights the feasibility of creating carriers with multiple functionalized CO-RMs.

Weight-reducing, lipid-lowering and antidiabetic activities of a novel arginine vasopressin analogue acting at the V1a and V1b receptors in high-fat-fed mice.

AIM: To assess the beneficial metabolic effects of the nonapeptide hormone, arginine vasopressin (AVP), on metabolism. MATERIALS AND METHODS: We exchanged amino acids at position 3 and 8 of AVP, namely phenylalanine and arginine, with those of oxytocin, to generate novel analogues with altered receptor selectivity. Secondary modification by N-terminal acetylation was used to impart stability to circulating endopeptidases. Analogues were screened for degradation, bioactivity in rodent/human clonal beta cells and primary murine islets, together with evaluation of receptor activation profile. RESULTS: Analogue Ac3IV, which lacked effects at the V2 receptors responsible for modulation of fluid balance, was selected as the lead compound for assessment of antidiabetic efficacy in high-fat-fed mice. Twice-daily administration of Ac3IV, or the gold standard control exendin-4, for 22 days, reduced energy intake as well as body weight and fat content. Both interventions decreased circulating glucose levels, enhanced insulin sensitivity, and substantially improved glucose tolerance and related insulin secretion in response to an intraperitoneal or oral glucose challenge. The peptides decreased total- and increased HDL-cholesterol, but only Ac3IV decreased LDL-cholesterol, triglyceride and non-fasting glucagon concentrations. Elevations of islet and beta-cell areas were partially reversed, accompanied by suppressed islet cell proliferation, decreased beta-cell apoptosis and, in the case of exendin-4, also decreased alpha-cell apoptosis. CONCLUSION: AVP-based therapies that exclusively target V1a and V1b receptors may have significant therapeutic potential for the treatment of obesity and related diabetes, and merit further clinical exploration.

An orally active carbon monoxide-releasing molecule enhances beneficial gut microbial species to combat obesity in mice.

Carbon monoxide (CO), a gaseous signaling molecule, has shown promise in preventing body weight gain and metabolic dysfunction induced by high fat diet (HFD), but the mechanisms underlying these effects are largely unknown. An essential component in response to HFD is the gut microbiome, which is significantly altered during obesity and represents a target for developing new therapeutic interventions to fight metabolic diseases. Here, we show that CO delivered to the gut by oral administration with a CO-releasing molecule (CORM-401) accumulates in faeces and enriches a variety of microbial species that were perturbed by a HFD regimen. Notably, Akkermansia muciniphila, which exerts salutary metabolic effects in mice and humans, was strongly depleted by HFD but was the most abundant gut species detected after CORM-401 treatment. Analysis of bacterial transcripts revealed a restoration of microbial functional activity, with partial or full recovery of the Krebs cycle, ß-oxidation, respiratory chain and glycolysis. Mice treated with CORM-401 exhibited normalization of several plasma and fecal metabolites that were disrupted by HFD and are dependent on Akkermansia muciniphila's metabolic activity, including indoles and tryptophan derivatives. Finally, CORM-401 treatment led to an improvement in gut morphology as well as reduction of inflammatory markers in colon and cecum and restoration of metabolic profiles in these tissues. Our findings provide therapeutic insights on the efficacy of CO as a potential prebiotic to combat obesity, identifying the gut microbiota as a crucial target for CO-mediated pharmacological activities against metabolic disorders.

Ac3IV, a V1a and V1b receptor selective vasopressin analogue, protects against hydrocortisone-induced changes in pancreatic islet cell lineage.

The Avpr1a (V1a) and Avpr1b (V1b) receptor selective, vasopressin (AVP) analogue, Ac3IV has been shown to improve metabolism and pancreatic islet structure in diabetes and insulin resistance. The present study further investigates these actions by assessing the ability of Ac3IV to protect against pancreatic islet architectural disturbances induced by hydrocortisone (HC) treatment in transgenic Ins1Cre/+;Rosa26-eYFP mice, that possess beta-cell lineage tracing capabilities. HC intervention increased (p < 0.001) energy intake but reduced (p < 0.01) body weight gain, with no impact of Ac3IV. All HC mice had reduced (p < 0.05) circulating glucose, but plasma insulin and glucagon concentrations remained unchanged. However, HC mice presented with increased (p < 0.001) pancreatic insulin content, which was further augmented by Ac3IV. In addition, Ac3IV treatment countered HC-induced increases in islet-, beta- and alpha-cell areas (p < 0.01), as well as promoting islet number towards control levels. This was accompanied by reduced (p < 0.05) beta-cell growth, but enhanced (p < 0.001) alpha-cell proliferation. There were no changes in islet cell apoptotic rates in any of the groups of HC mice, but co-expression of CK19 with insulin in pancreatic ductal cells was reduced by Ac3IV. Assessment of beta-cell lineage revealed that Ac3IV partially protected against HC-mediated de-differentiation of mature beta-cells, whilst also decreasing (p < 0.01) beta- to alpha-cell transdifferentiation. Our data indicate that sustained activation of V1a and V1b receptors exerts positive islet cell transition effects to help retain beta-cell identity in HC mice.

Beneficial impact of Ac3IV, an AVP analogue acting specifically at V1a and V1b receptors, on diabetes islet morphology and transdifferentiation of alpha- and beta-cells.

Ac3IV (Ac-CYIQNCPRG-NH2) is an enzymatically stable vasopressin analogue that selectively activates Avpr1a (V1a) and Avpr1b (V1b) receptors. In the current study we have employed streptozotocin (STZ) diabetic transgenic Ins1Cre/+;Rosa26-eYFP and GluCreERT2;Rosa26-eYFP mice, to evaluate the impact of sustained Ac3IV treatment on pancreatic islet cell morphology and transdifferentiation. Twice-daily administration of Ac3IV (25 nmol/kg bw) to STZ-diabetic Ins1Cre/+;Rosa26-eYFP mice for 12 days increased pancreatic insulin (p<0.01) and significantly reversed the detrimental effects of STZ on pancreatic islet morphology. Such benefits were coupled with increased (p<0.01) beta-cell proliferation and decreased (p<0.05) beta-cell apoptosis. In terms of islet cell lineage tracing, induction of diabetes increased (p<0.001) beta- to alpha-cell differentiation in Ins1Cre/+;Rosa26-eYFP mice, with Ac3IV partially reversing (p<0.05) such transition events. Comparable benefits of Ac3IV on pancreatic islet architecture were observed in STZ-diabetic GluCreERT2;ROSA26-eYFP transgenic mice. In this model, Ac3IV provoked improvements in islet morphology which were linked to increased (p<0.05-p<0.01) transition of alpha- to beta-cells. Ac3IV also increased (p<0.05-p<0.01) CK-19 co-expression with insulin in pancreatic ductal and islet cells. Blood glucose levels were unchanged by Ac3IV in both models, reflecting the severity of diabetes induced. Taken together these data indicate that activation of islet receptors for V1a and V1b positively modulates alpha- and beta-cell turnover and endocrine cell lineage transition events to preserve beta-cell identity and islet architecture.

Development and characterisation of novel, enzymatically stable oxytocin analogues with beneficial antidiabetic effects in high fat fed mice.

BACKGROUND: There is growing evidence to support beneficial effects of the hypothalamic synthesised hormone, oxytocin, on metabolism. However, the biological half-life of oxytocin is short and receptor activation profile unspecific. METHODS: We have characterised peptide-based oxytocin analogues with structural modifications aimed at improving half-life and receptor specificity. Following extensive in vitro and in vivo characterisation, antidiabetic efficacy of lead peptides was examined in high fat fed (HFF) mice. RESULTS: Following assessment of stability against enzymatic degradation, insulin secretory activity, receptor activation profile and in vivo bioactivity, analogues 2 N (Ac-C ˂YIQNC >PLG-NH2) and D7R ((d-C)YIQNCYLG-NH2) were selected as lead peptides. Twice daily injection of either peptide for 22 days reduced body weight, energy intake, plasma glucose and insulin and pancreatic glucagon content in HFF mice. In addition, both peptides reduced total- and LDL-cholesterol, with concomitant elevations of HDL-cholesterol, and D7R also decreased triglyceride levels. The two oxytocin analogues improved glucose tolerance and insulin responses to intraperitoneal, and particularly oral, glucose challenge on day 22. Both oxytocin analogues enhanced insulin sensitivity, reduced HOMA-IR and increased bone mineral density. In terms of pancreatic islet histology, D7R reversed high fat feeding induced elevations of islet and beta cell areas, which was associated with reductions in beta cell apoptosis. Islet insulin secretory responsiveness was improved by 2 N, and especially D7R, treatment. CONCLUSION: Novel, enzymatically stable oxytocin analogues exert beneficial antidiabetic effects in HFF mice. GENERAL SIGNIFICANCE: These observations emphasise the, yet untapped, therapeutic potential of long-acting oxytocin-based agents for obesity and type 2 diabetes.

Vasopressin receptors in islets enhance glucose tolerance, pancreatic beta-cell secretory function, proliferation and survival.

Arginine vasopressin (AVP), a peptide secreted from the posterior pituitary, is chiefly regarded as a hormone involved in the regulation of body fluid balance and osmolality. However, recent evidence has revealed that posterior pituitary hormones can exert important actions on endocrine pancreatic function. In the present study, the presence of AVP receptors, namely Avpr1a (V1a), Avpr1b (V1b) and Avpr2 (V2) was demonstrated in murine islets as well as rodent BRIN BD11 and human 1.1B4 beta-cells. Further to this, AVP was shown to induce significant concentration-dependent (10-12 - 10-6 M) increases of insulin release from both rodent and human beta-cells, as well as mouse islets. Insulinotropic actions of AVP were completely annulled by specific V1a or V1b receptor antagonists, and partially abolished by an oxytocin receptor antagonist. In addition, beta-cell insulin secretory actions of AVP were augmented by both IBMX (200 µM) and KCl (30 mM) and linked to significantly increased cAMP production and [Ca2+]i. AVP substantially increased proliferation of rodent and human beta-cells. Moreover, AVP fully protected against cytokine-induced beta-cell apoptosis. AVP had no effect on glucagon secretion. Immunohistochemical examination of beta- and alpha-cells revealed co-expression of AVP with glucagon, and particularly insulin. Finally, administration of AVP in combination with glucose to mice significantly reduced blood glucose, which was associated with increased plasma insulin. These data indicate that AVP possesses novel and potentially important effects on pancreatic endocrine function. Understanding disturbances in islet AVP receptor signalling could reveal insight into the beta-cell defects associated with diabetes.

Oxytocin is present in islets and plays a role in beta-cell function and survival.

Oxytocin is associated mainly with modulating reproductive function. However, studies suggest that oxytocin also plays a role in endocrine pancreatic function. In the present study, islet expression of oxytocin and its related receptor was confirmed in mouse islets as well as cultured rodent and human beta-cells. Oxytocin significantly stimulated glucose-induced insulin secretion from isolated mouse islets. Similar insulinotropic actions were also observed in rodent BRIN BD11 and human 1.1B4 beta-cells. Positive effects of oxytocin on insulin secretion were almost fully annulled by the oxytocin receptor antagonist, atosiban. In terms of mechanism of insulin secretory action, oxytocin had no effect on beta-cell membrane potential or cAMP generation, but did augment intracellular calcium concentrations. In vivo administration of oxytocin to mice significantly reduced overall blood glucose levels and increased plasma insulin concentrations in response to a glucose challenge. Oxytocin also had a modest, but significant, appetite suppressive effect. As expected, streptozotocin diabetic mice had marked loss of beta-cell area accompanied by increases in alpha-cell area, whilst hydrocortisone treatment increased beta-cell and overall islet areas. Both mouse models of diabetes presented with dramatically decreased percentage islet oxytocin co-localisation with insulin and increased co-localisation with glucagon. More detailed studies in cultured beta-cell lines revealed direct positive effects of oxytocin on beta-cell proliferation and protection against apoptosis. Together, these data highlight a potentially important role of islet-derived oxytocin and related receptor signalling pathways on the modulation of beta-cell function and survival.

Diagnosis of abnormal uterine bleeding.

Abnormal uterine bleeding is an extremely common indication for referral to a gynaecologist. This chapter examines the modes of presentation and the causes of such symptoms, which range from physiological variations to more sinister underlying pathology. A thorough understanding of these causes is required to direct investigation in an appropriate manner. The full range of possible investigations is discussed with emphasis on how to choose the most appropriate tests for a particular patient. This is fundamental to ensure that tests are pertinent and streamlined, and to prevent unnecessary anxiety and delay. Once the underlying causes have been clarified, a suitable management plan can be made.

Cervical screening in England and Wales: an update.

PURPOSE OF REVIEW: The National Health Service Cervical Screening Programme (NHSCSP) has played a major role in reducing the mortality from cervical cancer in England and Wales. However, the current system has numerous shortcomings and it is likely that its success has reached a plateau. In light of this, significant changes have recently been made to the programme. These alterations, as well as further potential developments, are considered here. RECENT FINDINGS: The aim of any change to the programme is to improve its sensitivity and specificity whilst reducing patient morbidity and maintaining cost-effectiveness. Alterations to NHSCSP guidelines include the replacement of the Papanicolau smear with liquid-based cytology, the referral for colposcopy of women with a single dyskaryotic cytology result and the commencement of screening at the later age of 25. These changes appear to be beneficial overall. The role of newer technologies in the programme is being clarified and it is likely that human papillomavirus testing will be incorporated in the near future. Progress is being made in the field of human papillomavirus vaccines, particularly prophylactic, which may go on to have the most profound impact on the incidence of cervical cancer. The disease is now largely a burden of the developing world, where the use of these technologies is considered. SUMMARY: Changes currently being instituted in the NHSCSP should go some way towards improving the service. The importance of increasing coverage rates, reducing patient waiting times and the associated anxiety must not be overlooked.