Effect of a Glp-1 Mimetic on the Insulin Response to Oral Sugar Testing in Horses

BACKGROUND: Insulin dysregulation (ID) is the most important risk factor for the development of laminitis in horses and therapies to control it are needed. HYPOTHESIS/OBJECTIVES: To assess the effects of a single dose of the synthetic GLP-1 analog exenatide on postprandial insulin dynamics. We hypothesized that exenatide would improve insulin sensitivity and lower postprandial blood insulin concentrations. STUDY DESIGN: Randomized, crossover, experimental study. ANIMALS: Six horses (3 mares, 3 geldings; 2 with normal insulin regulation [NIR] and 4 with mild ID). METHODS: Horses completed both study arms: subcutaneous administration of exenatide (or no treatment) 30 min before an oral sugar test (0.15 ml/kg of Karo Syrup). Blood samples obtained over 240 min were assayed for glucose, insulin, lactate, c-peptide and total GLP-1. The area under the curve (AUC) was calculated using the trapezoidal rule. Insulin sensitivity (S RESULTS: Exenatide resulted in a postprandial decrease of 20% (effect size: 2673 µU·min/ml; 95% CI: 900 - 4446 µU·min/ml; P = 0.003) in AUC of plasma insulin (control; mean AUC insulin: 11,989 µU·min/ml; 95% CI: 9673 - 14,305 µU·min/ml, exenatide; mean AUC insulin: 9316 µU·min/ml; 95% CI: 7430 - 11,202 µU·min/ml). Exenatide resulted in an approximately threefold increase (effect size: 5.56 10 CONCLUSIONS: The decrease in insulin response to carbohydrates was due to an increase in whole-body insulin sensitivity. GLP-1 agonists may have therapeutic potential for ID in horses

Gi- and Gs-coupled GPCRs show different modes of G-protein binding.

More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand-receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, Gs, and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR-G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin-G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, Gi, using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin-Gi complex, Gi engages rhodopsin in a manner distinct from previous GPCR-Gs structures, providing insight into specificity determinants

A novel model to assess lamellar signaling relevant to preferential weight bearing in the horse

Supporting limb laminitis (SLL) is a devastating sequela to severe unilateral lameness in equine patients. The manifestation of SLL, which usually only affects one limb, is unpredictable and the etiology is unknown. A novel, non-painful preferential weight bearing model designed to mimic the effects of severe unilateral forelimb lameness was developed to assess lamellar signaling events in the supporting limb (SL). A custom v-shaped insert was attached to the shoe of one forelimb to prevent normal weight bearing and redistribute weight onto the SL. Testing of the insert using a custom scale platform built into the floor of stocks confirmed increased distribution of weight on the SL compared with the unloaded forelimb (UL) and the contralateral (CH) and ipsilateral (IH) hind limbs in six Standardbred horses. In a second part of the study, eight healthy Standardbred horses were fitted with the insert and tied with consistent monitoring and free access to hay and water for 48 h, after which the lamellae were harvested. Real-time qPCR was performed to assess lamellar mRNA concentrations of inflammatory genes and immunoblotting and immunofluorescence were performed to assess lamellar protein concentration and cellular localization of hypoxia-related proteins, respectively. Lamellar mRNA concentrations of inflammatory signaling proteins did not differ between SL and either CH or IH samples. HIF-1α concentrations were greater (P

The effect of hypothermia on influx of leukocytes in the digital lamellae of horses with oligofructose-induced laminitis

Sepsis-related laminitis (SRL) is a common complication in the septic/endotoxemic critically-ill equine patient, in which lamellar injury and failure commonly lead to crippling distal displacement of the distal phalanx. Similar to organ injury in human sepsis, lamellar injury in SRL has been associated with inflammatory events, including the influx of leukocytes into the lamellar tissue and markedly increased expression of a wide array of inflammatory mediators at the onset of Obel grade 1 (OG1) laminitis. The only treatment reported both clinically and experimentally to protect the lamellae in SRL, local hypothermia (“cryotherapy”), has been demonstrated to effectively inhibit lamellar expression of multiple inflammatory mediators when initiated at the time of administration of a carbohydrate overload in experimental models of SRL. However, the effect of hypothermia on leukocyte influx into affected tissue has not been assessed. We hypothesized that cryotherapy inhibits leukocyte emigration into the digital lamellae in SRL. Immunohistochemical staining using leukocyte markers MAC387 (marker of neutrophils, activated monocytes) and CD163 (monocyte/macrophage-specific marker) was performed on archived lamellar tissue samples from an experimental model of SRL in which one forelimb was maintained at ambient temperature (AMB) and one forelimb was immersed in ice water (ICE) immediately following enteral oligofructose administration (10\ua0g/kg, n\ua0=\ua014 horses). Lamellae were harvested at 24\ua0h post-oligofructose administration (DEV, n\ua0=\ua07) or at the onset of OG1 laminitis (OG1, n\ua0=\ua07). Both MAC387-positive and CD163-positive cells were counted by a single blinded investigator on images [n\ua0=\ua010 (40× fields/digit for MAC387 and 20\ua0x fields/digit for CD163)] obtained using Aperio microscopy imaging analysis software. Data were assessed for normality and analyzed with a paired t-test and one-way ANOVA with significance set at p\ua

A liquid chromatography-tandem mass spectrometry-based investigation of the lamellar interstitial metabolome in healthy horses and during experimental laminitis induction

Lamellar bioenergetic failure is thought to contribute to laminitis pathogenesis but current knowledge of lamellar bioenergetic physiology is limited. Metabolomic analysis (MA) can systematically profile multiple metabolites. Applied to lamellar microdialysis samples (dialysate), lamellar bioenergetic changes during laminitis (the laminitis metabolome) can be characterised. The objectives of this study were to develop a technique for targeted MA of lamellar and skin dialysates in normal horses, and to compare the lamellar and plasma metabolomic profiles of normal horses with those from horses developing experimentally induced laminitis. Archived lamellar and skin dialysates (n = 7) and tissues (n = 6) from normal horses, and lamellar dialysate and plasma from horses given either 10 g/kg oligofructose (treatment group, OFT; n = 4) or sham (control group, CON; n = 4) were analysed. The concentrations of 44 intermediates of central carbon metabolism (CCM) were determined using liquid chromatography-tandem mass spectrometry. Data were analysed using multivariate (MVA) and univariate (UVA) analysis methods.The plasma metabolome appeared to be more variable than the lamellar metabolome by MVA, driven by malate, pyruvate, aconitate and glycolate. In lamellar dialysate, these metabolites decreased in OFT horses at the later time points. Plasma malate was markedly increased after 6 h in OFT horses. Plasma malate concentrations between OFT and CON at this time point were significantly different by UVA. MA of lamellar CCM was capable of differentiating horses developing experimental laminitis from controls. Lamellar malate, pyruvate, aconitate and glycolate, and plasma malate alone were identified as the source of differentiation between OFT and CON groups. These results highlighted clear discriminators between OFT and CON horses, suggesting that changes in energy metabolism occur locally in the lamellar tissue during laminitis development. The biological significance of these alterations requires further investigation

Equine lamellar energy metabolism studied using tissue microdialysis

Failure of lamellar energy metabolism may contribute to the pathophysiology of equine laminitis. Tissue microdialysis has the potential to dynamically monitor lamellar energy balance over time. The objectives of this study were to develop a minimally invasive lamellar microdialysis technique and use it to measure normal lamellar energy metabolite concentrations over 24h. Microdialysis probes were placed (through the white line) into either the lamellar dermis (LAM) (n=6) or the sublamellar dermis (SUBLAM) (n=6) and perfused continuously over a 24h study period. Probes were placed in the skin dermis (SKIN) for simultaneous comparison to LAM (n=6). Samples were collected every 2h and analysed for glucose, lactate, pyruvate, urea and glycerol concentrations. LAM was further compared with SUBLAM by simultaneous placement and sampling in four feet from two horses over 4h. Horses were monitored for lameness, and either clinically evaluated for 1month after probe removal (n=4) or subjected to histological evaluation of the probe site (n=10).There were no deleterious clinical effects of probe placement and the histological response was mild. Sample fluid recovery and metabolite concentrations were stable for 24h. Glucose was lower (and lactate:glucose ratio higher) in LAM compared with SUBLAM and SKIN (

Glomerular filtration rate is superior to serum creatinine for prediction of mortality after thoracoabdominal aortic surgery

BackgroundClinically evident renal disease (dialysis, history of renal insufficiency, or serum creatinine >2.0 mg/dL) is a known risk factor for mortality after thoracoabdominal aortic aneurysm repair. We extended this concept to the questions of whether subclinical renal disease is also a risk factor and how best to identify subclinical disease. We hypothesized that the glomerular filtration rate (GFR) would be a more sensitive determinant of renal function than serum creatinine alone.MethodsBetween 1991 and 2004, we repaired 1106 thoracoabdominal aortic aneurysms and descending thoracic aortic aneurysms. The median age was 67 years. There were 400 (36%) women and 706 (64%) men. We estimated GFR by using the Cockcroft-Gault equation. We divided baseline serum creatinine and baseline GFR into quartiles and estimated the association of the quartiles with 30-day postoperative mortality by χ2 testing. We further subdivided the population into patients with and without clinically evident renal disease and repeated the analysis in the patients without clinically apparent disease (n = 869).ResultsClinically apparent renal disease was highly associated with 30-day mortality (odds ratio, 3.2; P < .0001). In all patients, serum creatinine quartile and GFR quartile were also both highly significantly associated with 30-day mortality (P < .0001). In patients without clinically apparent renal disease, both creatinine and GFR predicted additional mortality, but GFR was a much stronger predictor (P < .02 for creatinine vs <.0001 for GFR). In these patients, mortality ranged from 5% in the best GFR quartile to 27% in the worst. Taken as continuous variables in logistic regression equations, serum creatinine had no discrimination in patients without clinical disease (P = .73), whereas GFR remained strong (P <.0001).ConclusionsPreoperative renal function is an important determinant of early mortality even in patients without clinically evident disease. Estimated GFR is a much more powerful determinant of mortality risk than serum creatinine alone

Beyond the 'Grid-Lock' in Electricity Interconnectors: The Case of Germany and Poland

The common European electricity market requires both market integration and transmission grid expansion, including trans-border interconnectors. Although the benefits of increased interconnectivity are widely acknowledged, expansion of interconnectors is often very slow. This paper gathers insights on the reasons behind this grid-lock drawing on the study of the German-Polish border. Although two interconnectors already exist, the trade is blocked by unplanned electricity loop flows. A third interconnector has been discussed for years, but saw little progress in spite of declarations of support on both sides. Drawing on the existing literature on the topic of grid expansion we identify four hypotheses for the grid-lock: inadequate financing; diverging interests; governance and administration problems; and different actors' motivations, trust and security perceptions. We evaluate them using the empirical material gathered through document analysis and stakeholder interviews conducted in Germany and Poland. None of the hypotheses on its own can explain the gridlock. However, while financing has not been a major obstacle, divergent interests had an impact on the project delay, administrative and governance problems are a great hindrance on the technical level, while motivations influence interstate political relations and policy shaping. EU support and closer bilateral cooperation provide opportunities to address these challenges

Continuous digital hypothermia prevents lamellar failure in the euglycaemic hyperinsulinaemic clamp model of equine laminitis

Continuous digital hypothermia can prevent the development and progression of laminitis associated with sepsis but its effects on laminitis due to hyperinsulinaemia are unknown.To determine the effects of continuous digital hypothermia on laminitis development in the euglycaemic hyperinsulinaemic clamp model.Randomised, controlled (within subject), blinded, experiment.Eight clinically normal Standardbred horses underwent laminitis induction using the euglycaemic hyperinsulinaemic clamp model (EHC). At initiation of the EHC, one forelimb was continuously cooled (ICE), with the other maintained at ambient temperature (AMB). Dorsal lamellar sections (proximal, middle, distal) were harvested 48 h after initiation of the EHC and were analysed using histological scoring (0-3) and histomorphometry. Cellular proliferation was quantified by counting epidermal cell nuclei staining positive with an immunohistochemical proliferation marker (TPX2).Severe elongation and disruption of SEL with dermo-epidermal separation (score of 3) was observed in all AMB feet at one or more section locations, but was not observed in any ICE sections. Overall 92% of AMB sections received the most severe histological score (grade 3) and 8% were grade 2, whereas ICE sections were classified as either grade 1 (50%) or grade 2 (50%). Relative to AMB feet, ICE sections were 98% less likely to exhibit grades 2 or 3 (OR: 0.02, 95% CI 0.001, 0.365;

10-Year Paclitaxel Dose-Related Outcomes of Drug-Eluting Stents Treated Below the Knee in Patients with Chronic Limb-Threatening Ischemia (The PADI Trial)

Purpose: Recently, two meta-analyses concluded that there appears to be an increased risk of long-term mortality of paclitaxel-coated balloons and stents in the superficial femoral and popliteal artery, and paclitaxel-coated balloons below the knee. In this post hoc study of the PADI Trial, we investigated the long-term safety of first-generation paclitaxel-coated drug-eluting stents (DES) below the knee and the dose–mortality relationships of paclitaxel in patients with chronic limb-threatening ischemia (CLI). Materials and Methods: The PADI Trial compared paclitaxel-coated DES with percutaneous transluminal angioplasty with bail-out bare-metal stents (PTA ± BMS) in patients with CLI treated below the knee. Follow-up was extended to 10 years after the first inclusion, and survival analyses were performed. In addition, dose-related mortality and dose per patient weight-related mortality relations were examined. Results: A total of 140 limbs in 137 patients were included in the PADI Trial. Ten years after the first inclusion, 109/137 (79.6%) patients had died. There was no significant difference between mortality in the DES group compared with the PTA ± BMS group (Log-rank p value = 0.12). No specific dose-related mortality (HR 1.00, 95% CI 0.99–1.00, p = 0.99) or dose per weight mortality (HR 1.05, 95% CI 0.93–1.18, p = 0.46) relationships were identified in the Cox-proportional Hazard models or by Kaplan–Meier survival analyses. Conclusions: There is a poor 10-year survival in both paclitaxel-coated DES and PTA ± BMS in patients with CLI treated below the knee. No dose-related adverse effects of paclitaxel-coated DES were observed in our study of patients with CLI treated below the knee. Level of Evidence: The PADI Trial: level 1, randomized clinical trial