Structure-Activity Relationships and Discovery of (S)-6-Isopropyl-2-methoxy-3-(3-methoxypropoxy)-10-oxo-5,10-dihydro-6H-pyrido[1,2-h][1,7]naphthyridine-9-carboxylic Acid (AB-452), a Novel Orally Available HBV RNA Destabilizer.

Approved therapies for hepatitis B virus (HBV) treatment include nucleos(t)ides and interferon alpha (IFN-α) which effectively suppress viral replication, but they rarely lead to cure. Expression of viral proteins, especially surface antigen of the hepatitis B virus (HBsAg) from covalently closed circular DNA (cccDNA) and the integrated genome, is believed to contribute to the persistence of HBV. This work focuses on therapies that target the expression of HBV proteins, in particular HBsAg, which differs from current treatments. Here we describe the identification of AB-452, a dihydroquinolizinone (DHQ) analogue. AB-452 is a potent HBV RNA destabilizer by inhibiting PAPD5/7 proteins in vitro with good in vivo efficacy in a chronic HBV mouse model. AB-452 showed acceptable tolerability in 28-day rat and dog toxicity studies, and a high degree of oral exposure in multiple species. Based on its in vitro and in vivo profiles, AB-452 was identified as a clinical development candidate.

Design, synthesis, and structure-activity relationship of a bicyclic HBV capsid assembly modulator chemotype leading to the identification of clinical candidate AB-506.

Disruption of the HBV capsid assembly process through small-molecule interaction with HBV core protein is a validated target for the suppression of hepatitis B viral replication and the development of new antivirals. Through combination of key structural features associated with two distinct series of capsid assembly modulators, a novel aminochroman-based chemotype was identified. Optimization of anti-HBV potency through generation of SAR in addition to further core modifications provided a series of related functionalized aminoindanes. Key compounds demonstrated excellent cellular potency in addition to favorable ADME and pharmacokinetic profiles and were shown to be highly efficacious in a mouse model of HBV replication. Aminoindane derivative AB-506 was subsequently advanced into clinical development.

The Rho guanine dissociation inhibitor α inhibits skeletal muscle Rac1 activity and insulin action.

The molecular events governing skeletal muscle glucose uptake have pharmacological potential for managing insulin resistance in conditions such as obesity, diabetes, and cancer. With no current pharmacological treatments to target skeletal muscle insulin sensitivity, there is an unmet need to identify the molecular mechanisms that control insulin sensitivity in skeletal muscle. Here, the Rho guanine dissociation inhibitor α (RhoGDIα) is identified as a point of control in the regulation of insulin sensitivity. In skeletal muscle cells, RhoGDIα interacted with, and thereby inhibited, the Rho GTPase Rac1. In response to insulin, RhoGDIα was phosphorylated at S101 and Rac1 dissociated from RhoGDIα to facilitate skeletal muscle GLUT4 translocation. Accordingly, siRNA-mediated RhoGDIα depletion increased Rac1 activity and elevated GLUT4 translocation. Consistent with RhoGDIα's inhibitory effect, rAAV-mediated RhoGDIα overexpression in mouse muscle decreased insulin-stimulated glucose uptake and was detrimental to whole-body glucose tolerance. Aligning with RhoGDIα's negative role in insulin sensitivity, RhoGDIα protein content was elevated in skeletal muscle from insulin-resistant patients with type 2 diabetes. These data identify RhoGDIα as a clinically relevant controller of skeletal muscle insulin sensitivity and whole-body glucose homeostasis, mechanistically by modulating Rac1 activity.

The identification of highly efficacious functionalised tetrahydrocyclopenta[c]pyrroles as inhibitors of HBV viral replication through modulation of HBV capsid assembly.

Disruption of the HBV viral life cycle with small molecules that prevent the encapsidation of pregenomic RNA and viral polymerase through binding to HBV core protein is a clinically validated approach to inhibiting HBV viral replication. Herein we report the further optimisation of clinical candidate AB-506 through core modification with a focus on increasing oral exposure and oral half-life. Maintenance of high levels of anti-HBV cellular potency in conjunction with improvements in pharmacokinetic properties led to multi-log10 reductions in serum HBV DNA following low, once-daily oral dosing for key analogues in a preclinical animal model of HBV replication.

Preclinical characterization of AB-506, an inhibitor of HBV replication targeting the viral core protein.

AB-506, a small-molecule inhibitor targeting the HBV core protein, inhibits viral replication in vitro (HepAD38 cells: EC50 of 0.077 µM, CC50 > 25 µM) and in vivo (HBV mouse model: ∼3.0 log10 reductions in serum HBV DNA compared to the vehicle control). Binding of AB-506 to HBV core protein accelerates capsid assembly and inhibits HBV pgRNA encapsidation. Furthermore, AB-506 blocks cccDNA establishment in HBV-infected HepG2-hNTCP-C4 cells and primary human hepatocytes, leading to inhibition of viral RNA, HBsAg, and HBeAg production (EC50 from 0.64 µM to 1.92 µM). AB-506 demonstrated activity across HBV genotypes A-H and maintains antiviral activity against nucleos(t)ide analog-resistant variants in vitro. Evaluation of AB-506 against a panel of core variants showed that T33N/Q substitutions results in >200-fold increase in EC50 values, while L30F, L37Q, and I105T substitutions showed an 8 to 20-fold increase in EC50 values in comparison to the wild-type. In vitro combinations of AB-506 with NAs or an RNAi agent were additive to moderately synergistic. AB-506 exhibits good oral bioavailability, systemic exposure, and higher liver to plasma ratios in rodents, a pharmacokinetic profile supporting clinical development for chronic hepatitis B.

HBsAg mRNA degradation induced by a dihydroquinolizinone compound depends on the HBV posttranscriptional regulatory element.

In pursuit of novel therapeutics targeting the hepatitis B virus (HBV) infection, we evaluated a dihydroquinolizinone compound (DHQ-1) that in the nanomolar range reduced the production of virion and surface protein (HBsAg) in tissue culture. This compound also showed broad HBV genotype coverage, but was inactive against a panel of DNA and RNA viruses of other species. Oral administration of DHQ-1 in the AAV-HBV mouse model resulted in a significant reduction of serum HBsAg as soon as 4 days following the commencement of treatment. Reduction of HBV markers in both in vitro and in vivo experiments was related to the reduced amount of viral RNA including pre-genomic RNA (pgRNA) and 2.4/2.1 kb HBsAg mRNA. Nuclear run-on and subcellular fractionation experiments indicated that DHQ-1 mediated HBV RNA reduction was the result of accelerated viral RNA degradation in the nucleus, rather than the consequence of inhibition of transcription initiation. Through mutagenesis of HBsAg gene sequences, we found induction of HBsAg mRNA decay by DHQ-1 required the presence of the HBV posttranscriptional regulatory element (HPRE), with a 109 nucleotides sequence within the central region of the HPRE alpha sub-element being the most critical. Taken together, the current study shows that a small molecule can reduce the overall levels of HBV RNA, especially the HBsAg mRNA, and viral surface proteins. This may shed light on the development of a new class of HBV therapeutics.

Signaling of the p21-activated kinase (PAK1) coordinates insulin-stimulated actin remodeling and glucose uptake in skeletal muscle cells.

Skeletal muscle accounts for ∼ 80% of postprandial glucose clearance, and skeletal muscle glucose clearance is crucial for maintaining insulin sensitivity and euglycemia. Insulin-stimulated glucose clearance/uptake entails recruitment of glucose transporter 4 (GLUT4) to the plasma membrane (PM) in a process that requires cortical F-actin remodeling; this process is dysregulated in Type 2 Diabetes. Recent studies have implicated PAK1 as a required element in GLUT4 recruitment in mouse skeletal muscle in vivo, although its underlying mechanism of action and requirement in glucose uptake remains undetermined. Toward this, we have employed the PAK1 inhibitor, IPA3, in studies using L6-GLUT4-myc muscle cells. IPA3 fully ablated insulin-stimulated GLUT4 translocation to the PM, corroborating the observation of ablated insulin-stimulated GLUT4 accumulation in the PM of skeletal muscle from PAK1(-/-) knockout mice. IPA3-treatment also abolished insulin-stimulated glucose uptake into skeletal myotubes. Mechanistically, live-cell imaging of myoblasts expressing the F-actin biosensor LifeAct-GFP treated with IPA3 showed blunting of the normal insulin-induced cortical actin remodeling. This blunting was underpinned by a loss of normal insulin-stimulated cofilin dephosphorylation in IPA3-treated myoblasts. These findings expand upon the existing model of actin remodeling in glucose uptake, by placing insulin-stimulated PAK1 signaling as a required upstream step to facilitate actin remodeling and subsequent cofilin dephosphorylation. Active, dephosphorylated cofilin then provides the G-actin substrate for continued F-actin remodeling to facilitate GLUT4 vesicle translocation for glucose uptake into the skeletal muscle cell.

Signal transduction meets vesicle traffic: the software and hardware of GLUT4 translocation.

Skeletal muscle is the major tissue disposing of dietary glucose, a function regulated by insulin-elicited signals that impart mobilization of GLUT4 glucose transporters to the plasma membrane. This phenomenon, also central to adipocyte biology, has been the subject of intense and productive research for decades. We focus on muscle cell studies scrutinizing insulin signals and vesicle traffic in a spatiotemporal manner. Using the analogy of an integrated circuit to approach the intersection between signal transduction and vesicle mobilization, we identify signaling relays ("software") that engage structural/mechanical elements ("hardware") to enact the rapid mobilization and incorporation of GLUT4 into the cell surface. We emphasize how insulin signal transduction switches from tyrosine through lipid and serine phosphorylation down to activation of small G proteins of the Rab and Rho families, describe key negative regulation step of Rab GTPases through the GTPase-activating protein activity of the Akt substrate of 160 kDa (AS160), and focus on the mechanical effectors engaged by Rabs 8A and 10 (the molecular motor myosin Va), and the Rho GTPase Rac1 (actin filament branching and severing through Arp2/3 and cofilin). Finally, we illustrate how actin filaments interact with myosin 1c and α-Actinin4 to promote vesicle tethering as preamble to fusion with the membrane.

Myosin Va mediates Rab8A-regulated GLUT4 vesicle exocytosis in insulin-stimulated muscle cells.

Rab-GTPases are important molecular switches regulating intracellular vesicle traffic, and we recently showed that Rab8A and Rab13 are activated by insulin in muscle to mobilize GLUT4-containing vesicles to the muscle cell surface. Here we show that the unconventional motor protein myosin Va (MyoVa) is an effector of Rab8A in this process. In CHO-IR cell lysates, a glutathione S-transferase chimera of the cargo-binding COOH tail (CT) of MyoVa binds Rab8A and the related Rab10, but not Rab13. Binding to Rab8A is stimulated by insulin in a phosphatidylinositol 3-kinase-dependent manner, whereas Rab10 binding is insulin insensitive. MyoVa-CT preferentially binds GTP-locked Rab8A. Full-length green fluorescent protein (GFP)-MyoVa colocalizes with mCherry-Rab8A in perinuclear small puncta, whereas GFP-MyoVa-CT collapses the GTPase into enlarged perinuclear depots. Further, GFP-MyoVa-CT blocks insulin-stimulated translocation of exofacially myc-tagged GLUT4 to the surface of muscle cells. Mutation of amino acids in MyoVa-CT predicted to bind Rab8A abrogates both interaction with Rab8A (not Rab10) and inhibition of insulin-stimulated GLUT4myc translocation. Of importance, small interfering RNA-mediated MyoVa silencing reduces insulin-stimulated GLUT4myc translocation. Rab8A colocalizes with GLUT4 in perinuclear but not submembrane regions visualized by confocal total internal reflection fluorescence microscopy. Hence insulin signaling to the molecular switch Rab8A connects with the motor protein MyoVa to mobilize GLUT4 vesicles toward the muscle cell plasma membrane.

Akt and Rac1 signaling are jointly required for insulin-stimulated glucose uptake in skeletal muscle and downregulated in insulin resistance.

Skeletal muscle plays a major role in regulating whole body glucose metabolism. Akt and Rac1 are important regulators of insulin-stimulated glucose uptake in skeletal muscle. However the relative role of each pathway and how they interact are not understood. Here we delineate how Akt and Rac1 pathways signal to increase glucose transport independently of each other and are simultaneously downregulated in insulin resistant muscle. Pharmacological inhibition of Rac1 and Akt signaling was used to determine the contribution of each pathway to insulin-stimulated glucose uptake in mouse muscles. The actin filament-depolymerizing agent LatrunculinB was combined with pharmacological inhibition of Rac1 or Akt, to examine whether either pathway mediates its effect via the actin cytoskeleton. Akt and Rac1 signaling were investigated under each condition, as well as upon Akt2 knockout and in ob/ob mice, to uncover whether Akt and Rac1 signaling are independent and whether they are affected by genetically-induced insulin resistance. While individual inhibition of Rac1 or Akt partially decreased insulin-stimulated glucose transport by ~40% and ~60%, respectively, their simultaneous inhibition completely blocked insulin-stimulated glucose transport. LatrunculinB plus Akt inhibition blocked insulin-stimulated glucose uptake, while LatrunculinB had no additive effect on Rac1 inhibition. In muscles from severely insulin-resistant ob/ob mice, Rac1 and Akt signaling were severely dysregulated and the increment in response to insulin reduced by 100% and 90%, respectively. These findings suggest that Rac1 and Akt regulate insulin-stimulated glucose uptake via distinct parallel pathways, and that insulin-induced Rac1 and Akt signaling are both dysfunctional in insulin resistant muscle. There may thus be multiple treatment targets for improving insulin sensitivity in muscle.

Rac-1 superactivation triggers insulin-independent glucose transporter 4 (GLUT4) translocation that bypasses signaling defects exerted by c-Jun N-terminal kinase (JNK)- and ceramide-induced insulin resistance.

Insulin activates a cascade of signaling molecules, including Rac-1, Akt, and AS160, to promote the net gain of glucose transporter 4 (GLUT4) at the plasma membrane of muscle cells. Interestingly, constitutively active Rac-1 expression results in a hormone-independent increase in surface GLUT4; however, the molecular mechanism and significance behind this effect remain unresolved. Using L6 myoblasts stably expressing myc-tagged GLUT4, we found that overexpression of constitutively active but not wild-type Rac-1 sufficed to drive GLUT4 translocation to the membrane of comparable magnitude with that elicited by insulin. Stimulation of endogenous Rac-1 by Tiam1 overexpression elicited a similar hormone-independent gain in surface GLUT4. This effect on GLUT4 traffic could also be reproduced by acutely activating a Rac-1 construct via rapamycin-mediated heterodimerization. Strategies triggering Rac-1 "superactivation" (i.e. to levels above those attained by insulin alone) produced a modest gain in plasma membrane phosphatidylinositol 3,4,5-trisphosphate, moderate Akt activation, and substantial AS160 phosphorylation, which translated into GLUT4 translocation and negated the requirement for IRS-1. This unique signaling capacity exerted by Rac-1 superactivation bypassed the defects imposed by JNK- and ceramide-induced insulin resistance and allowed full and partial restoration of the GLUT4 translocation response, respectively. We propose that potent elevation of Rac-1 activation alone suffices to drive insulin-independent GLUT4 translocation in muscle cells, and such a strategy might be exploited to bypass signaling defects during insulin resistance.

Rac1 is a novel regulator of contraction-stimulated glucose uptake in skeletal muscle.

In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (~60-100%) and humans (~40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20-58% in extensor digitorum longus (EDL; P < 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P < 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P < 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake.

Muscle cells challenged with saturated fatty acids mount an autonomous inflammatory response that activates macrophages.

Obesity is associated with chronic low-grade inflammation. Within adipose tissue of mice fed a high fat diet, resident and infiltrating macrophages assume a pro-inflammatory phenotype characterized by the production of cytokines which in turn impact on the surrounding tissue. However, inflammation is not restricted to adipose tissue and high fat-feeding is responsible for a significant increase in pro-inflammatory cytokine expression in muscle. Although skeletal muscle is the major disposer of dietary glucose and a major determinant of glycemia, the origin and consequence of muscle inflammation in the development of insulin resistance are poorly understood.We used a cell culture approach to investigate the vectorial crosstalk between muscle cells and macrophages upon exposure to physiological, low levels of saturated and unsaturated fatty acids. Inflammatory pathway activation and cytokine expression were analyzed in L6 muscle cells expressing myc-tagged GLUT4 (L6GLUT4myc) exposed to 0.2 mM palmitate or palmitoleate. Conditioned media thereof, free of fatty acids, were then tested for their ability to activate RAW264.7 macrophages.Palmitate -but not palmitoleate- induced IL-6, TNFα and CCL2 expression in muscle cells, through activation of the NF-κB pathway. Palmitate (0.2 mM) alone did not induce insulin resistance in muscle cells, yet conditioned media from palmitate-challenged muscle cells selectively activated macrophages towards a pro-inflammatory phenotype.These results demonstrate that low concentrations of palmitate activate autonomous inflammation in muscle cells to release factors that turn macrophages pro-inflammatory. We hypothesize that saturated fat-induced, low-grade muscle cell inflammation may trigger resident skeletal muscle macrophage polarization, possibly contributing to insulin resistance in vivo.

Evaluation of phosphatidylinositol-4-kinase IIIα as a hepatitis C virus drug target.

Phosphatidylinositol-4-kinase IIIα (PI4KIIIα) is an essential host cell factor for hepatitis C virus (HCV) replication. An N-terminally truncated 130-kDa form was used to reconstitute an in vitro biochemical lipid kinase assay that was optimized for small-molecule compound screening and identified potent and specific inhibitors. Cell culture studies with PI4KIIIα inhibitors demonstrated that the kinase activity was essential for HCV RNA replication. Two PI4KIIIα inhibitors were used to select cell lines harboring HCV replicon mutants with a 20-fold loss in sensitivity to the compounds. Reverse genetic mapping isolated an NS4B-NS5A segment that rescued HCV RNA replication in PIK4IIIα-deficient cells. HCV RNA replication occurs on specialized membranous webs, and this study with PIK4IIIα inhibitor-resistant mutants provides a genetic link between NS4B/NS5A functions and PI4-phosphate lipid metabolism. A comprehensive assessment of PI4KIIIα as a drug target included its evaluation for pharmacologic intervention in vivo through conditional transgenic murine lines that mimic target-specific inhibition in adult mice. Homozygotes that induce a knockout of the kinase domain or knock in a single amino acid substitution, kinase-defective PI4KIIIα, displayed a lethal phenotype with a fairly widespread mucosal epithelial degeneration of the gastrointestinal tract. This essential host physiologic role raises doubt about the pursuit of PI4KIIIα inhibitors for treatment of chronic HCV infection.

Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles.

GLUT4-containing vesicles cycle between the plasma membrane and intracellular compartments. Insulin promotes GLUT4 exocytosis by regulating GLUT4 vesicle arrival at the cell periphery and its subsequent tethering, docking, and fusion with the plasma membrane. The molecular machinery involved in GLUT4 vesicle tethering is unknown. We show here that Myo1c, an actin-based motor protein that associates with membranes and actin filaments, is required for insulin-induced vesicle tethering in muscle cells. Myo1c was found to associate with both mobile and tethered GLUT4 vesicles and to be required for vesicle capture in the total internal reflection fluorescence (TIRF) zone beneath the plasma membrane. Myo1c knockdown or overexpression of an actin binding-deficient Myo1c mutant abolished insulin-induced vesicle immobilization, increased GLUT4 vesicle velocity in the TIRF zone, and prevented their externalization. Conversely, Myo1c overexpression immobilized GLUT4 vesicles in the TIRF zone and promoted insulin-induced GLUT4 exposure to the extracellular milieu. Myo1c also contributed to insulin-dependent actin filament remodeling. Thus we propose that interaction of vesicular Myo1c with cortical actin filaments is required for insulin-mediated tethering of GLUT4 vesicles and for efficient GLUT4 surface delivery in muscle cells.

Rac1 signalling towards GLUT4/glucose uptake in skeletal muscle.

Small Rho family GTPases are important regulators of cellular traffic. Emerging evidence now implicates Rac1 and Rac-dependent actin reorganisation in insulin-induced recruitment of glucose transporter-4 (GLUT4) to the cell surface of muscle cells and mature skeletal muscle. This review summarises the current thinking on the regulation of Rac1 by insulin, the role of Rac-dependent cortical actin remodelling in GLUT4 traffic, and the impact of Rac1 towards insulin resistance in skeletal muscle.

NOD2 activation induces muscle cell-autonomous innate immune responses and insulin resistance.

Insulin resistance is associated with chronic low-grade inflammation in vivo, largely mediated by activated innate immune cells. Cytokines and pathogen-derived ligands of surface toll-like receptors can directly cause insulin resistance in muscle cells. However, it is not known if intracellular pathogen sensors can, on their own, provoke insulin resistance. Here, we show that the cytosolic pattern recognition receptors nucleotide-binding oligomerization domain-containing protein (NOD)1 and NOD2 are expressed in immune and metabolic tissues and hypothesize that their activation in muscle cells would result in cell-autonomous responses leading to insulin resistance. Bacterial peptidoglycan motifs that selectively activate NOD2 were directly administered to L6- GLUT4myc myotubes in culture. Within 3 h, insulin resistance arose, characterized by reductions in each insulin-stimulated glucose uptake, GLUT4 translocation, Akt Ser(473) phosphorylation, and insulin receptor substrate 1 tyrosine phosphorylation. Muscle cell-autonomous responses to NOD2 ligand included activation of the stress/inflammation markers c-Jun N-terminal kinase, ERK1/2, p38 MAPK, degradation of inhibitor of κBα, and production of proinflammatory cytokines. These results show that NOD2 alone is capable of acutely inducing insulin resistance within muscle cells, possibly by activating endogenous inflammatory signals and/or through cytokine production, curbing upstream insulin signals. NOD2 is hence a new inflammation target connected to insulin resistance, and this link occurs without the need of additional contributing cell types. This study provides supporting evidence for the integration of innate immune and metabolic responses through the involvement of NOD proteins and suggests the possible participation of cell autonomous immune responses in the development of insulin resistance in skeletal muscle, the major depot for postprandial glucose utilization.

Arp2/3- and cofilin-coordinated actin dynamics is required for insulin-mediated GLUT4 translocation to the surface of muscle cells.

GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.