Calcineurin regulates skeletal muscle metabolism via coordinated changes in gene expression

The metabolic property of skeletal muscle adapts in response to an increased physiological demand by altering substrate utilization and gene expression. The calcium-regulated serine/threonine protein phosphatase calcineurin has been implicated in the transduction of motor neuron signals to alter gene expression programs in skeletal muscle. We utilized transgenic mice that overexpress an activated form of calcineurin in skeletal muscle (MCK-CnA*) to investigate the impact of calcineurin activation on metabolic properties of skeletal muscle. Activation of calcineurin increased glucose incorporation into glycogen and lipid oxidation in skeletal muscle. Activated calcineurin suppressed skeletal muscle glucose oxidation and increased lactate release. The enhancement in lipid oxidation was supported by increased expression of genes for lipid metabolism and mitochondrial oxidative phosphorylation. In a reciprocal fashion, several genes of glycolysis were down-regulated, whereas pyruvate dehydrogenase kinase 4 was markedly induced. This expression pattern was associated with decreased glucose utilization and enhanced glycogen storage. The peroxisome proliferator-activated receptors (PPARs) and PPARgamma coactivator 1alpha (PGC1alpha) are transcription regulators for the expression of metabolic and mitochondrial genes. Consistent with changes in the gene-regulatory program, calcineurin promoted the expression of PPARalpha, PPARdelta, and PPARgamma coactivator 1alpha in skeletal muscle. These results provide evidence that calcineurin-mediated skeletal muscle reprogramming induces the expression of several transcription regulators that coordinate changes in the expression of genes for lipid and glucose metabolism, which in turn alters energy substrate utilization in skeletal muscle

Increased hepatic insulin sensitivity in mice lacking inhibitory leptin receptor signals

Leptin regulates food intake and energy expenditure by activating the long form of the leptin receptor (LepRb). Leptin also regulates glucose homeostasis by improving whole-body insulin sensitivity, but the mechanism remains undefined. Leptin action is mediated by phosphorylation of several tyrosine residues on LepRb. LepRb-Tyr985 plays an important role in the attenuation of LepRb signaling. We determined the contribution of LepRb-Tyr985-mediated signals to leptin action on insulin sensitivity using LepRb-Tyr985 mutant mice (l/l mice). Glucose tolerance and whole-body insulin-mediated glucose utilization were determined in wild-type (+/+) and l/l mice. Glucose tolerance was unaltered between female +/+ and l/l mice but enhanced in the male l/l mice. Serum insulin concentration was decreased at baseline and 15 min after a glucose injection in female l/l vs. +/+ mice (P < 0.05) but unaltered in the male l/l mice. However, basal and insulin-stimulated glucose transport in isolated soleus and extensor digitorum longus muscle was similar between +/+ and l/l mice, indicating skeletal muscle insulin sensitivity in vitro was not enhanced. Moreover, euglycemic-hyperinsulinemic clamps reveal hepatic, rather than peripheral, insulin sensitivity is enhanced in female l/l mice, whereas male l/l mice display both improved hepatic and peripheral insulin sensitivity. In conclusion, signals emanating from leptin receptor Tyr985 control hepatic insulin sensitivity in both female and male l/l mice. Lack of LepRb-Tyr985 signaling enhances whole-body insulin sensitivity partly through increased insulin action on the suppression of hepatic glucose production

Opposite transcriptional regulation in skeletal muscle of AMP-activated protein kinase gamma3 R225Q transgenic versus knock-out mice

AMP-activated protein kinase (AMPK) is an evolutionarily conserved heterotrimer important for metabolic sensing in all eukaryotes. The muscle-specific isoform of the regulatory gamma-subunit of the kinase, AMPK gamma3, has an important role in glucose uptake, glycogen synthesis, and fat oxidation in white skeletal muscle, as previously demonstrated by physiological characterization of AMPK gamma3 mutant (R225Q) transgenic (TgPrkag3(225Q)) and gamma3 knock-out (Prkag3(-/-)) mice. We determined AMPK gamma3-dependent regulation of gene expression by analyzing global transcription profiles in glycolytic skeletal muscle from gamma3 mutant transgenic and knock-out mice using oligonucleotide microarray technology. Evidence is provided for coordinated and reciprocal regulation of multiple key components in glucose and fat metabolism, as well as skeletal muscle ergogenics in TgPrkag3(225Q) and Prkag3(-/-) mice. The differential gene expression profile was consistent with the physiological differences between the models, providing a molecular mechanism for the observed phenotype. The striking pattern of opposing transcriptional changes between TgPrkag3(225Q) and Prkag3(-/-) mice identifies differentially expressed targets being truly regulated by AMPK and is consistent with the view that R225Q is an activating mutation, in terms of its downstream effects. Additionally, we identified a wide array of novel targets and regulatory pathways for AMPK in skeletal muscle

Neuregulins mediate calcium-induced glucose transport during muscle contraction

Neuregulin, a growth factor involved in myogenesis, has rapid effects on muscle metabolism. In a manner analogous to insulin and exercise, neuregulins stimulate glucose transport through recruitment of glucose transporters to surface membranes in skeletal muscle. Like muscle contraction, neuregulins have additive effects with insulin on glucose uptake. Therefore, we examined whether neuregulins are involved in the mechanism by which muscle contraction regulates glucose transport. We show that caffeine-induced increases in cytosolic Ca2+ mediate a metalloproteinase-dependent release of neuregulins, which stimulates tyrosine phosphorylation of ErbB4 receptors. Activation of ErbB4 is necessary for Ca2+-derived effects on glucose transport. Furthermore, blockage of ErbB4 abruptly impairs contraction-induced glucose uptake in slow twitch muscle fibers, and to a lesser extent, in fast twitch muscle fibers. In conclusion, we provide evidence that contraction-induced activation of neuregulin receptors is necessary for the stimulation of glucose transport and a key element of energetic metabolism during muscle contraction

Idarucizumab for Dabigatran Reversal - Full Cohort Analysis.

BACKGROUND: Idarucizumab, a monoclonal antibody fragment, was developed to reverse the anticoagulant effect of dabigatran. METHODS: We performed a multicenter, prospective, open-label study to determine whether 5 g of intravenous idarucizumab would be able to reverse the anticoagulant effect of dabigatran in patients who had uncontrolled bleeding (group A) or were about to undergo an urgent procedure (group B). The primary end point was the maximum percentage reversal of the anticoagulant effect of dabigatran within 4 hours after the administration of idarucizumab, on the basis of the diluted thrombin time or ecarin clotting time. Secondary end points included the restoration of hemostasis and safety measures. RESULTS: A total of 503 patients were enrolled: 301 in group A, and 202 in group B. The median maximum percentage reversal of dabigatran was 100% (95% confidence interval, 100 to 100), on the basis of either the diluted thrombin time or the ecarin clotting time. In group A, 137 patients (45.5%) presented with gastrointestinal bleeding and 98 (32.6%) presented with intracranial hemorrhage; among the patients who could be assessed, the median time to the cessation of bleeding was 2.5 hours. In group B, the median time to the initiation of the intended procedure was 1.6 hours; periprocedural hemostasis was assessed as normal in 93.4% of the patients, mildly abnormal in 5.1%, and moderately abnormal in 1.5%. At 90 days, thrombotic events had occurred in 6.3% of the patients in group A and in 7.4% in group B, and the mortality rate was 18.8% and 18.9%, respectively. There were no serious adverse safety signals. CONCLUSIONS: In emergency situations, idarucizumab rapidly, durably, and safely reversed the anticoagulant effect of dabigatran. (Funded by Boehringer Ingelheim; RE-VERSE AD ClinicalTrials.gov number, NCT02104947 .)

Molecular mechanisms governing contraction-induced metabolic responses and skeletal muscle reprogramming

Physical exercise enhances skeletal muscle responsiveness to insulin and regulates metabolism by an insulin-independent mechanism. Elucidation of contraction-mediated molecular mechanisms is imperative for a better understanding of skeletal muscle metabolism and function, and may lead to the identification or validation of possible drug targets for the prevention or treatment of metabolic disorders. This thesis focuses on the role of AMPK and Interleukin (IL)-6 in skeletal muscle metabolism, because AMPK activity and skeletal muscle IL-6 release are increased during skeletal muscle contraction. Contraction-mediated AMPK activity in white glycolytic extensor digitorum longus (EDL) muscle was inversely coupled to skeletal muscle glycogen content in wild-type and transgenic mice expressing a mutated form of the AMPKgamma3 isoform (Tg-Prkag3225Q), but not AMPKgamma3 knockout (KO) mice, highlighting a role for the AMPKgamma3 isoform in energy sensing during muscle contraction. Isolated skeletal muscle from Tg-Prkag3225Q and AMPKgamma3 KO mice were fatigue-resistant and fatigue-prone, respectively; and work performance was coupled to glycogen content in all mouse models, highlighting a role for AMPKgamma3 in skeletal muscle ergogenics by controlling glycogen levels. Hypoxia-mediated glucose transport was partly reduced in skeletal muscle from AMPKgamma3 KO mice. AICAR and contraction-mediated phosphorylation of the Akt substrate, AS160, was dependent on functional AMPK signaling, providing direct genetic evidence that AS160 is a phosphorylation target of AMPK. IL-6 is released from contracting human skeletal muscle. We provide evidence that IL-6 release is greater from oxidative, compared to glycolytic skeletal muscle. Basal IL-6 release was increased from oxidative soleus muscle of AMPKá1 KO and AMPKá2 kinasedead mice. Thus, we provide evidence for a role of AMPK in the basal regulation of IL-6 release from isolated oxidative skeletal muscle. Furthermore, AICAR-mediated suppression of basal IL-6 mRNA production and release was independent of functional AMPK signaling. Autocrine mechanisms may play a role in basal IL-6 release from isolated skeletal muscle. IL-6 concentrations in the contracting skeletal muscle may exceed serum levels. We therefore investigated the direct effect of IL-6 on human skeletal muscle by exposing primary human skeletal muscle cells and isolated human skeletal muscle strips to IL-6. IL-6-exposure directly influences glucose metabolism, as determined by increased glucose transport and glucose incorporation into glycogen. In primary human skeletal muscle cells, IL-6-exposure activated components of the canonical insulin signaling cascade. Glucose incorporation into glycogen was sensitive to phosphatidylinositol (PI) 3-kinase inhibition. In contrast, IL-6-exposure was without effect on insulin signaling in isolated human skeletal muscle, and increased glucose metabolism was observed, concomitant with a trend for increased phosphorylation of AMPK. In primary human muscle cells, the IL-6-mediated enhancement of fatty acid oxidation was attenuated by silencing AMPKá isoforms. Long-term IL-6-exposure of primary myotubes enhanced growth and differentiation and increased the expression of genes involved in muscle metabolism. In conclusion, this thesis work provides evidence that AMPK and IL-6 are central players in the regulation of contraction-mediated effects on metabolic responses in skeletal muscle. In addition to the involvement in acute regulation of metabolism, IL-6 may also participate in skeletal muscle adaptation to exercise