Intracellular signaling pathways of muscarinic acetylcholine receptor-mediated detrusor muscle contractions.

Acetylcholine plays an essential role in the regulation of detrusor muscle contractions, and antimuscarinics are widely used in the management of overactive bladder syndrome. However, several adverse effects limit their application and patients' compliance. Thus, this study aimed to further analyze the signal transduction of M2 and M3 receptors in the murine urinary bladder to eventually find more specific therapeutic targets. Experiments were performed on adult male wild-type, M2, M3, M2/M3, or Gαq/11 knockout (KO), and pertussis toxin (PTX)-treated mice. Contraction force and RhoA activity were measured in the urinary bladder smooth muscle (UBSM). Our results indicate that carbamoylcholine (CCh)-induced contractions were associated with increased activity of RhoA and were reduced in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632 in UBSM. CCh-evoked contractile responses and RhoA activation were markedly reduced in detrusor strips lacking either M2 or M3 receptors and abolished in M2/M3 KO mice. Inhibition of Gαi-coupled signaling by PTX treatment shifted the concentration-response curve of CCh to the right and diminished RhoA activation. CCh-induced contractile responses were markedly decreased in Gαq/11 KO mice; however, RhoA activation was unaffected. In conclusion, cholinergic detrusor contraction and RhoA activation are mediated by both M2 and M3 receptors. Furthermore, whereas both Gαi and Gαq/11 proteins mediate UBSM contraction, the activation at the RhoA-ROCK pathway appears to be linked specifically to Gαi. These findings may aid the identification of more specific therapeutic targets for bladder dysfunctions.NEW & NOTEWORTHY Muscarinic acetylcholine receptors are of utmost importance in physiological regulation of micturition and also in the development of voiding disorders. We demonstrate that the RhoA-Rho-associated kinase (ROCK) pathway plays a crucial role in contractions induced by cholinergic stimulation in detrusor muscle. Activation of RhoA is mediated by both M2 and M3 receptors as well as by Gi but not Gq/11 proteins. The Gi-RhoA-ROCK pathway may provide a novel therapeutic target for overactive voiding disorders.

Benefits of insulin degludec/liraglutide are maintained even in patients discontinuing sulphonylureas or dipeptidyl peptidase-4 inhibitors upon initiation of degludec/liraglutide therapy: A post hoc analysis of the DUAL II and DUAL IX trials.

AIM: To investigate the efficacy and safety of initiating insulin degludec/liraglutide (IDegLira) in patients with type 2 diabetes (T2D) who had discontinued pretrial sulphonylureas (SUs) or dipeptidyl peptidase-4 inhibitors (DPP4is) versus patients not previously treated with these regimens. MATERIALS AND METHODS: In DUAL II, patients with T2D uncontrolled on basal insulin and metformin ± SU/glinides were randomized to insulin degludec or IDegLira (both capped at 50 U). In DUAL IX, patients were randomized to insulin glargine U100 (no maximum dose) or IDegLira, as add-on to sodium-glucose co-transporter-2 inhibitors ± oral antidiabetic drugs. In this post hoc analysis, patients were grouped according to pretrial use of SU (DUAL II) or DPP4i (DUAL IX). RESULTS: Regardless of pretrial SU/DPP4i use, IDegLira was favourable versus insulin comparators with respect to change in HbA1c and body weight. Lower hypoglycaemia rates and comparable end-of-trial daily insulin dose were achieved with IDegLira, regardless of pretrial regimen. There was no clinically relevant increase in mean self-measured blood glucose in the early weeks after IDegLira initiation. There was no statistically significant interaction between the randomized treatments and previous SU/DPP4i use. CONCLUSIONS: IDegLira was more favourable compared with degludec or glargine U100 in terms of change in HbA1c and body weight, regardless of antecedent treatment. Clinicians should be aware of a potential transient rise in self-measured blood glucose when transitioning therapy in patients. This shows that SUs/DPP4is can be safely discontinued, without deterioration in glycaemic control when initiating IDegLira, allowing a simplified treatment regimen.

A greater proportion of participants with type 2 diabetes achieve treatment targets with insulin degludec/liraglutide versus insulin glargine 100 units/mL at 26 weeks: DUAL VIII, a randomized trial designed to resemble clinical practice.

This report presents the efficacy and safety of insulin degludec/liraglutide (IDegLira) versus insulin glargine 100 units/mL (IGlar U100) as initial injectable therapy at 26 weeks in the 104-week DUAL VIII durability trial (NCT02501161). Participants (N = 1012) with type 2 diabetes (T2D) uncontrolled on oral antidiabetic drugs (OADs) were randomized 1:1 to open-label IDegLira or IGlar U100. Visits were scheduled at weeks 1, 2, 4 and 12, and every 3 months thereafter. After 26 weeks, glycated haemoglobin (HbA1c) reductions were greater with IDegLira versus IGlar U100 (-21.5 vs. -16.4 mmol/mol [-2.0 vs. -1.5%]), as was the percentage of participants achieving HbA1c <53 mmol/mol (78.7% vs. 55.7%) and HbA1c targets without weight gain and/or hypoglycaemia. Estimated treatment differences for insulin dose (-13.01 U) and body weight change (-1.57 kg) significantly favoured IDegLira. The hypoglycaemia rate was 44% lower with IDegLira versus IGlar U100. Safety results were similar. In a trial resembling clinical practice, more participants receiving IDegLira than IGlar U100 met treatment targets, supporting use of IDegLira as an initial injectable therapy for people with T2D uncontrolled on OADs and eligible for insulin initiation.

NK2 receptor-mediated detrusor muscle contraction involves Gq/11-dependent activation of voltage-dependent Ca2+ channels and the RhoA-Rho kinase pathway.

Tachykinins (TKs) are involved in both the physiological regulation of urinary bladder functions and development of overactive bladder syndrome. The aim of the present study was to investigate the signal transduction pathways of TKs in the detrusor muscle to provide potential pharmacological targets for the treatment of bladder dysfunctions related to enhanced TK production. Contraction force, intracellular Ca2+ concentration, and RhoA activity were measured in the mouse urinary bladder smooth muscle (UBSM). TKs and the NK2 receptor (NK2R)-specific agonist [ß-Ala8]-NKA(4-10) evoked contraction, which was inhibited by the NKR2 antagonist MEN10376. In Gαq/11-deficient mice, [ß-Ala8]-NKA(4-10)-induced contraction and the intracellular Ca2+ concentration increase were abolished. Although Gq/11 proteins are linked principally to phospholipase Cß and inositol trisphosphate-mediated Ca2+ release from intracellular stores, we found that phospholipase Cß inhibition and sarcoplasmic reticulum Ca2+ depletion failed to have any effect on contraction induced by [ß-Ala8]-NKA(4-10). In contrast, lack of extracellular Ca2+ or blockade of voltage-dependent Ca2+ channels (VDCCs) suppressed contraction. Furthermore, [ß-Ala8]-NKA(4-10) increased RhoA activity in the UBSM in a Gq/11-dependent manner and inhibition of Rho kinase with Y-27632 decreased contraction force, whereas the combination of Y-27632 with either VDCC blockade or depletion of extracellular Ca2+ resulted in complete inhibition of [ß-Ala8]-NKA(4-10)-induced contractions. In summary, our results indicate that NK2Rs are linked exclusively to Gq/11 proteins in the UBSM and that the intracellular signaling involves the simultaneous activation of VDCC and the RhoA-Rho kinase pathway. These findings may help to identify potential therapeutic targets of bladder dysfunctions related to upregulation of TKs.

IDegLira improves patient-reported outcomes while using a simple regimen with fewer injections and dose adjustments compared with basal-bolus therapy.

AIMS: Basal-bolus therapy is associated with greater treatment burden and lower adherence compared with more simplified regimens. This post hoc analysis studied the difference between insulin degludec/liraglutide (IDegLira) and basal-bolus therapy on number of injections, dose adjustments and patient outcomes in the DUAL VII trial. MATERIALS AND METHODS: DUAL VII was a 26-week, open-label trial in which patients with uncontrolled type 2 diabetes who were using metformin and insulin glargine 100 units/mL (20-50 U) were randomized 1:1 to IDegLira (N = 252) or basal-bolus (insulin glargine U100 + insulin aspart ≤4 times/day) (N = 254). This post hoc analysis reports the observed mean number of injections and cumulative dose adjustments during 26 weeks of treatment. Patient-reported outcomes (Treatment-Related Impact Measure - Diabetes [TRIM-D] and Short Form-36 Health Survey version 2 [SF-36v2]) were collected at scheduled visits and change from baseline scores calculated. RESULTS: The clinical benefits (non-inferior HbA1c reductions, weight benefit, less hypoglycaemia) of IDegLira vs basal-bolus therapy were achieved with fewer cumulative dose adjustments (16.6 vs 217.2, respectively) and fewer injections (1 vs ≥3 per day, respectively). Patients treated with IDegLira experienced significant improvements across all TRIM-D domains compared with those undergoing basal-bolus therapy. The SF-36v2 showed improvements in both treatment arms with no significant difference between arms in the physical component summary, but there was a significant improvement in patients treated with IDegLira in the mental component summary (P = .0228). CONCLUSIONS: These findings, combined with the DUAL VII results, suggest that IDegLira, through a more simplified regimen versus basal-bolus therapy, may help improve patient adherence and improve patient outcomes related to diabetes management, treatment burden and mental health, which in turn may assist in the timely achievement of glycaemic control in clinical practice.

Lower rates of cardiovascular events and mortality associated with liraglutide use in patients treated with basal insulin: A DEVOTE subanalysis (DEVOTE 10).

AIM: To compare the associations between concomitant liraglutide use versus no liraglutide use and the risk of major adverse cardiovascular events (MACE) and all-cause mortality among patients receiving basal insulin (either insulin degludec [degludec] or insulin glargine 100 units/mL [glargine U100]) in the Trial Comparing Cardiovascular Safety of Insulin Degludec versus Insulin Glargine in Patients with Type 2 Diabetes at High Risk of Cardiovascular Events (DEVOTE). MATERIALS AND METHODS: Patients with type 2 diabetes and high cardiovascular risk were randomized 1:1 to degludec or glargine U100. Hazard ratios for MACE/mortality were calculated using a Cox regression model adjusted for treatment and time-varying liraglutide use at any time during the trial, without interaction. Sensitivity analyses were adjusted for baseline covariates including, but not limited to, age, sex, smoking and prior cardiovascular disease. RESULTS: At baseline, 436/7637 (5.7%) patients were treated with liraglutide; after baseline, 187/7637 (2.4%) started and 210/7637 (2.7%) stopped liraglutide. Mean liraglutide exposure from randomization was 530.2 days. Liraglutide use versus no liraglutide use was associated with significantly lower hazard rates for MACE [0.62 (0.41; 0.92)95%CI ] and all-cause mortality [0.50 (0.29; 0.88)95%CI ]. There was no significant difference in the rate of severe hypoglycaemia with versus without liraglutide use. Multiple sensitivity analyses yielded similar results. CONCLUSIONS: Use of liraglutide was associated with significantly lower risk of MACE and death in patients with type 2 diabetes and high cardiovascular risk using basal insulin.

Elevated systemic TGF-beta impairs aortic vasomotor function through activation of NADPH oxidase-driven superoxide production and leads to hypertension, myocardial remodeling, and increased plaque formation in apoE(-/-) mice.

The role of circulating, systemic TGF-beta levels in endothelial function is not clear. TGF-beta(1) may cause endothelial dysfunction in apolipoprotein E-deficient (apoE(-/-)) mice via stimulation of reactive oxygen species (ROS) production by the NADPH oxidase (NOX) system and aggravate aortic and heart remodeling and hypertension. Thoracic aorta (TA) were isolated from 4-mo-old control (C57Bl/6), apoE(-/-), TGF-beta(1)-overexpressing (TGFbeta(1)), and crossbred apoE(-/-) x TGFbeta(1) mice. Endothelium-dependent relaxation was measured before and after incubation with apocynin (NOX inhibitor) or superoxide dismutase (SOD; ROS scavenger). Superoxide production within the vessel wall was determined by dihydroethidine staining under confocal microscope. In 8-mo-old mice, aortic and myocardial morphometric changes, plaque formation by en face fat staining, and blood pressure were determined. Serum TGF-beta(1) levels (ELISA) were elevated in TGFbeta(1) mice without downregulation of TGF-beta-I receptor (immunohistochemistry). In the aortic wall, superoxide production was enhanced and NO-dependent relaxation diminished in apoE(-/-) x TGFbeta(1) mice but improved significantly after apocynin or SOD. Myocardial capillary density was reduced, fibrocyte density increased, aortic wall was thicker, combined lesion area was greater, and blood pressure was higher in the apoE(-/-) x TGFbeta vs. C57Bl/6 mice. Our results demonstrate that elevated circulating TGF-beta(1) causes endothelial dysfunction through NOX activation-induced oxidative stress, accelerating atherosclerosis and hypertension in apoE(-/-) mice. These findings may provide a mechanism explaining accelerated atherosclerosis in patients with elevated plasma TGFbeta(1).

Durability of insulin degludec plus liraglutide versus insulin glargine U100 as initial injectable therapy in type 2 diabetes (DUAL VIII): a multicentre, open-label, phase 3b, randomised controlled trial.

BACKGROUND: Durability of glycaemic control might reduce disease burden and improve long-term outcomes. DUAL VIII investigated the durability of insulin degludec plus liraglutide (IDegLira) versus insulin glargine 100 units/mL (IGlar U100) in patients with type 2 diabetes with the use of a visit schedule that mirrored routine clinical practice. METHODS: In this 104-week international, multicentre, open-label, phase 3b randomised controlled trial, insulin-naive patients aged 18 years and older, with HbA1c between 7·0-11·0% (53-97 mmol/mol), BMI of 20 kg/m2 or higher, on stable doses of oral antidiabetic drugs, were recruited from outpatient clinics. Patients were randomly assigned 1:1, with a simple sequential allocation randomisation schedule (block size of four), to IDegLira or IGlar U100, each treatment being an add-on to existing therapy. The internal safety committee, the independent external committee, and the personnel involved in defining the analysis sets were masked until the database was released for statistical analysis. Patients and all other investigators were not masked. In the IDegLira group, patients were given degludec 100 units/mL plus liraglutide 3·6 mg/mL in a 3 mL prefilled PDS290 pen for subcutaneous injection; in the IGlar U100 group, patients were given IGlar U100 solution, in a 3 mL prefilled Solostar pen for subcutaneous injection. Both treatments were given once daily at any time of day and it was recommended that the time of day remained the same throughout the trial. The primary endpoint was time from randomisation to need for treatment intensification (HbA1c ≥7·0% [53 mmol/mol] at two consecutive visits, including week 26). Once patients met this criterion, the trial product was permanently discontinued and patients were not withdrawn from trial but rather remained on follow-up for the entire treatment and follow-up period. The primary analysis was in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT02501161. FINDINGS: From Jan 8, 2016, to Oct 3, 2018, 1345 patients were screened, of which 1012 (75·2%) were eligible and randomly assigned to either IDegLira (n=506) or IGlar U100 (n=506). 484 (96%) of 506 in the IDegLira group and 481 (95%) of 506 in the IGlar U100 group completed the trial. Baseline characteristics were similar and representative of patients eligible for basal insulin intensification (overall mean diabetes duration 10 years; HbA1c 8·5% [69 mmol/mol]; fasting plasma glucose 10 mmol/L). Patients in the IDegLira group had significantly longer time until intensification was needed than those in the IGlar U100 group (median >2 years vs about 1 year). Fewer patients in the IDegLira group needed treatment intensification over 104 weeks than those in the IGlar U100 group (189 [37%] of 506 vs 335 [66%] of 506). The preplanned sensitivity analyses of the primary endpoint were in agreement with the primary analysis (hazard ratio 0·45 [95% CI 0·38-0·54]) in the proportional hazards regression model and the generalised log-rank test was also in favour of IDegLira (p<0·0001). No new or unexpected safety and tolerability issues were identified and there were no treatment-related deaths. INTERPRETATION: In patients with uncontrolled type 2 diabetes on oral antidiabetic drugs, initial injectable therapy with IDegLira resulted in fewer patients reaching the treatment intensification criterion during 104 weeks versus IGlar U100, with longer durability of the treatment effect with IDegLira. FUNDING: Novo Nordisk.

G12-G13-LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension.

The tone of vascular smooth muscle cells is a primary determinant of the total peripheral vascular resistance and hence the arterial blood pressure. Most forms of hypertension ultimately result from an increased vascular tone that leads to an elevated total peripheral resistance. Regulation of vascular resistance under normotensive and hypertensive conditions involves multiple mediators, many of which act through G protein-coupled receptors on vascular smooth muscle cells. Receptors that mediate vasoconstriction couple with the G-proteins G(q)-G11 and G12-G13 to stimulate phosphorylation of myosin light chain (MLC) via the Ca2+/MLC kinase- and Rho/Rho kinase-mediated signaling pathways, respectively. Using genetically altered mouse models that allow for the acute abrogation of both signaling pathways by inducible Cre/loxP-mediated mutagenesis in smooth muscle cells, we show that G(q)-G11-mediated signaling in smooth muscle cells is required for maintenance of basal blood pressure and for the development of salt-induced hypertension. In contrast, lack of G12-G13, as well as of their major effector, the leukemia-associated Rho guanine nucleotide exchange factor (LARG), did not alter normal blood pressure regulation but did block the development of salt-induced hypertension. This identifies the G12-G13-LARG-mediated signaling pathway as a new target for antihypertensive therapies that would be expected to leave normal blood pressure regulation unaffected.

Endothelial NOS-mediated relaxations of isolated thoracic aorta of the C57BL/6J mouse: a methodological study.

Endothelium-dependent relaxations were studied in the thoracic aorta (TA) of the C57BL/6J mouse, a strain used commonly in the generation of genetically altered mice, to clarify some methodological questions. First, we have tested if transcardial perfusion with heparinized Krebs solution before the preparation of the TA may improve in vitro relaxant responses. Carbachol, thrombin, and ATP induced significantly stronger relaxations in TAs prepared from perfused animals than in controls. The effect of sodium nitroprusside (SNP), however, did not change, indicating that the improvement of the endothelium-dependent relaxations after perfusion was not caused by increased reactivity of the vascular smooth muscle to NO. Second, the potential regional differences within the TA were studied. Carbachol relaxed significantly stronger distal than proximal TA segments, whereas the effects of thrombin, ATP, and SNP showed no regional heterogeneity. Third, the relaxant effect of carbachol was partially preserved in TAs of endothelial NOS deficient (eNOS-/-) animals and remained unchanged in the presence of indomethacin, indicating the involvement of an eNOS- and cyclooxygenase-independent mechanism in the mediation of the response. Thrombin and ATP were ineffective in eNOS-/- TAs. Finally, TAs prepared from mice housed in cages equipped with running wheels did not show improved reactivity, indicating that the conventional housing conditions and the consequent sedentary lifestyle of the laboratory mouse do not diminish endothelial function in the TA.