Abstract: Conventional (c) protein kinase C (PKC) activity has been shown to increase with skeletal muscle contraction, and numerous studies using primarily pharmacological inhibitors have implicated cPKCs in contraction-stimulated glucose uptake. Here, to confirm that cPKC activity is required for contraction-stimulated glucose uptake in mouse muscles, contraction-stimulated glucose uptake ex vivo was first evaluated in the presence of three commonly used cPKC inhibitors (calphostin C, Gö-6976, and Gö-6983) in incubated mouse soleus and extensor digitorum longus (EDL) muscles. All potently inhibited contraction-stimulated glucose uptake by 50–100%, whereas both Gö compounds, but not calphostin C, inhibited insulin-stimulated glucose uptake modestly. AMP-activated protein kinase (AMPK) and eukaryotic elongation factor 2 phosphorylation was unaffected by the blockers. PKCα was estimated to account for ∼97% of total cPKC protein expression in skeletal muscle. However, in muscles from PKCα knockout (KO) mice, neither contraction- nor phorbol ester-stimulated glucose uptake ex vivo differed compared with the wild type. Furthermore, the effects of calphostin C and Gö-6983 on contraction-induced glucose uptake were similar in muscles lacking PKCα and in the wild type. It can be concluded that PKCα, representing ∼97% of cPKC in skeletal muscle, is not required for contraction-stimulated glucose uptake. Thus the effect of the PKC blockers on glucose uptake is either nonspecific working on other parts of contraction-induced signaling or the remaining cPKC isoforms are sufficient for stimulating glucose uptake during contractions.

Abstract: Some studies suggest that the 5′-AMP-activated protein kinase (AMPK) is important in regulating muscle glucose uptake in response to intense electrically stimulated contractions. However, it is unknown whether AMPK regulates muscle glucose uptake during in vivo exercise. We studied this in male and female mice overexpressing kinase-dead AMPKα2 (AMPK-KD) in skeletal and heart muscles. Wild-type and AMPK-KD mice were exercised at the same absolute intensity and the same relative intensity (30 and 70% of individual maximal running speed) to correct for reduced exercise capacity of the AMPK-KD mouse. Muscle glucose clearance was measured using 2-deoxy-[ 3 H]glucose as tracer. In wild-type mice, glucose clearance was increased at 30 and 70% of maximal running speed by 40 and 350% in the quadriceps muscle and by 120 and 380% in gastrocnemius muscle, respectively. Glucose clearance was not lower in AMPK-KD muscles compared with wild-type regardless of whether animals were exercised at the same relative or the same absolute intensity. In agreement, surface membrane content of the glucose transporter GLUT4 was increased similarly in AMPK-KD and wild-type muscle in response to running. We also measured signaling of alternative exercise-sensitive pathways that might be compensatorily increased in AMPK-KD muscles. However, increases in phosphorylation of CaMKII, Trisk95, p38 MAPK, and ERK1/2 were not higher in AMPK-KD than in WT muscle. Collectively, these findings suggest that AMPKα2 signaling is not essential in regulating glucose uptake in mouse skeletal muscle during treadmill exercise and that other mechanisms play a central role.

Abstract: Sea level change is an important indicator of climate change. Our study focuses on the sea level budget assessment of the Arctic Ocean using: (1) the newly reprocessed satellite altimeter data with major changes in the processing techniques; (2) ocean mass change data derived from GRACE satellite gravimetry; (3) and steric height estimated from gridded hydrographic data for the GRACE/Argo time period (2003–2016). The Beaufort Gyre (BG) and the Nordic Seas (NS) regions exhibit the largest positive trend in sea level during the study period. Halosteric sea level change is found to dominate the area averaged sea level trend of BG, while the trend in NS is found to be influenced by halosteric and ocean mass change effects. Temporal variability of sea level in these two regions reveals a significant shift in the trend pattern centered around 2009–2011. Analysis suggests that this shift can be explained by a change in large-scale atmospheric circulation patterns over the Arctic. The sea level budget assessment of the Arctic found a residual trend of more than 1.0 mm/yr. This nonclosure of the sea level budget is further attributed to the limitations of the three above mentioned datasets in the Arctic region.

Abstract: Abstract. A new mean sea surface (MSS) from the Technical University of Denmark (DTU) called DTU21MSS for referencing sea-level anomalies from satellite altimetry is introduced in this paper, and a suite of evaluations are performed. One of the reasons for updating the existing mean sea surface is the fact that during the last 6 years, nearly 3 times as many data have been made available by space agencies, resulting in more than 15 years of altimetry from long-repeat orbits (LROs) or geodetic missions. This includes the two interleaved long-repeat cycles of Jason-2 with a systematic cross-track distance as low as 4 km. A new processing chain with updated filtering and editing has been implemented for the DTU21MSS. This way, the DTU21MSS has been computed from 2 Hz altimetry in contrast to the former DTU15MSS and DTU18MSS which were computed from 1 Hz altimetry. The new DTU21MSS is computed over the same 20-year averaging time from 1 January 1993 to 31 December 2012 with a well-specified central time of 1 January 2003 and is available from https://doi.org/10.11583/DTU.19383221.v1 (Andersen, 2022). Cryosat-2 employs synthetic aperture radar (SAR) and SAR interferometric (SARin) modes in a large part of the Arctic Ocean due to the presence of sea ice. For SAR- and SARin-mode data we applied the SAMOSA+ physical retracking to make it compatible with the physical retracker used for conventional low-resolution-mode data in other parts of the ocean.

Abstract: Abstract. The Arctic Ocean is at the frontier of the fast-changing climate in the northern latitudes, and sea level trends are a bulk measure of ongoing processes related to climate change. Observations of sea level in the Arctic Ocean are nonetheless difficult to validate with independent measurements, and this is globally the region where the sea level trend (SLT) is most uncertain. The aim of this study is to create a satellite-independent reconstruction of Arctic SLT, as it is observed by altimetry and tide gauges (TGs). Previous studies use Gravity Recovery and Climate Experiment (GRACE) observations to estimate the manometric (mass component of) SLT. GRACE estimates, however, are challenged by large mass changes on land, which are difficult to separate from much smaller ocean mass changes. Furthermore, GRACE is not available before 2003, which significantly limits the period and makes the trend more vulnerable to short-term changes. As an alternative approach, this study estimates the climate-change-driven Arctic manometric SLT from the Arctic sea level fingerprints of glaciers, Greenland, Antarctica and glacial isostatic adjustment (GIA) with the addition of the long-term inverse barometer (IB) effect. The halosteric and thermosteric components complete the reconstructed Arctic SLT and are estimated by interpolating 300 000 temperature (T) and salinity (S) in situ observations. The SLT from 1995–2015 is compared to the observed SLT from altimetry and 12 selected tide gauges (TGs) corrected for vertical land movement (VLM). The reconstructed estimate manifests the salinity-driven halosteric component as dominating the spatial SLT pattern with variations between −7 and 10 mm yr−1. The manometric SLT in comparison is estimated to be 1–2 mm yr−1 for most of the Arctic Ocean. The reconstructed SLT shows a larger sea level rise in the Beaufort Sea compared to altimetry, an issue that is also identified by previous studies. There is a TG-observed sea level rise in the Siberian Arctic in contrast to the sea level fall from the reconstructed and altimetric estimate. From 1995–2015 the reconstructed SLT agrees within the 68 % confidence interval with the SLT from observed altimetry in 87 % of the Arctic between 65∘ N and 82∘ N (R=0.50) and with 5 of 12 TG-derived (VLM-corrected) SLT estimates. The residuals are seemingly smaller than results from previous studies using GRACE estimates and modeled T–S data. The spatial correlation of the reconstructed SLT to altimetric SLT during the GRACE period (2003–2015) is R=0.38 and R=0.34/R=0.37 if GRACE estimates are used instead of the constructed manometric component. Thus, the reconstructed manometric component is suggested as a legitimate alternative to GRACE that can be projected into the past and future.

Abstract: The treatment of hemophilia A (HA) is primarily based on replacement of factor VIII (FVIII), and in people with HA with inhibitors (HAwI) on the use of by-passing agents. Recently, a FVIII mimetic bispecific antibody emicizumab (Hemlibra®) was approved for treatment of HA and HAwI, offering a subcutaneous, prophylactic treatment opportunity with potential for significantly reducing the treatment burden. We describe the development and pre-clinical characterization of Mim8, a novel, next-generation FVIII mimetic human bispecific antibody. Mim8 is a highly potent molecule bridging factor IXa (FIXa) and factor X (FX) in development for subcutaneous treatment of people with HA and HAwI. Development of Mim8 utilized the Duobody® platform to initially screen for compatible anti-FIXa and anti-FX antibodies followed by several iterations of systematic mutational optimization. In total, more than 30,000 bispecific antibodies were analyzed. The optimization process aimed for efficient Mim8-mediated activation of FX by FIXa in the presence of procoagulant membrane, low target binding in solution, low immunogenicity risk, and for desirable biophysical parameters such as low viscosity. In vitro characterization demonstrated that Mim8 efficiently localizes FIXa and FX to the phospholipid surface and enhances FXa activation. The monovalent anti-FIXa arm alone stimulates the proteolytic activity of FIXa in the range of 15,000-fold and is an important contributor to the activity of the bispecific antibody. The dissociation constants (Kd) of Mim8 for FIXa and FX is in the micromolar range, minimizing target binding in the blood. Using thrombin generation assay in congenital HA plasma and thrombelastography (TEG) in whole blood from healthy volunteers spiked with anti-FVIII antibodies, Mim8 was capable of normalizing thrombin generation and blood clot formation, respectively, with approximately 15 times greater potency than emicizumab (Figure 1). A similar potency improvement was demonstrated in a tail vein transection bleeding model in FVIII-deficient mice co-dosed with human FIX and FX to circumvent lack of Mim8 cross reactivity to murine FIX and FX. The terminal half-life of Mim8 was estimated to 14 days (range 10-17 days) in cynomolgus monkeys and the subcutaneous bioavailability to 97%. In conclusion, Mim8 is a novel, next-generation FVIII mimetic bispecific antibody with anti-FIXa and anti-FX arms that potently stimulates FX activation resulting in efficacious haemostasis in vitro and in vivo. Mim8 has a high potency allowing for administration of small volumes in a pen device, good PK parameters, minimal target binding in the blood, and good biophysical properties. Collectively, these properties support clinical development of Mim8 as a potentially improved next-generation FVIII-mimetic prophylactic treatment option for persons with hemophilia A regardless of inhibitor status. Figure 1: Left: FXI-triggered thrombin generation assay in congenital HA plasma (mean and SD of n = 5). Right: thromboelastography in whole blood from healthy donors spiked with polyclonal anti-FVIII antibody (mean and SD of n = 3). Coagulation was triggered with low concentration (∼30 fM) of tissue factor (Innovin® 1:200,000). Shaded areas: standard deviation of controls. Blue circles: Mim8. Grey squares: a sequence identical analogue (SIA) to emicizumab (comparable data were obtained with a commercially available batch of Hemlibra®). Disclosures Kjellev: Novo Nordisk A/S: Employment, Equity Ownership. Østergaard:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Greisen:Novo Nordisk A/S: Equity Ownership, Patents & Royalties: Patents. Hermit:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Thorn:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Hansen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Zhou:Novo Nordisk A/S: Equity Ownership, Other: Previous employment, Patents & Royalties: Patents. Bjelke:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Kjalke:Novo Nordisk A/S: Employment, Honoraria. Lund:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Holm:Novo Nordisk A/S: Equity Ownership, Other: Previous employment. Ley:Novo Nordisk A/S: Employment, Equity Ownership. Elenius:Novo Nordisk A/S: Equity Ownership, Other: Previous employment; Leo Pharma A/S: Employment, Equity Ownership. Thygesen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Loftager:Novo Nordisk A/S: Employment, Equity Ownership. Rasch:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Lorenzen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Gandhi:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Lamberth:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Egebjerg:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Lund:Novo Nordisk A/S: Employment, Equity Ownership. Henriksen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Rahbek-Nielsen:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Yang:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties: Patents. Hilden:Novo Nordisk A/S: Employment, Equity Ownership, Patents & Royalties.