Palmitate-induced insulin resistance causes actin filament stiffness and GLUT4 mis-sorting without altered Akt signalling.

Skeletal muscle insulin resistance, a major contributor to type 2 diabetes, is linked to the consumption of saturated fats. This insulin resistance arises from failure of insulin-induced translocation of glucose transporter type 4 (GLUT4; also known as SLC2A4) to the plasma membrane to facilitate glucose uptake into muscle. The mechanisms of defective GLUT4 translocation are poorly understood, limiting development of insulin-sensitizing therapies targeting muscle glucose uptake. Although many studies have identified early insulin signalling defects and suggest that they are responsible for insulin resistance, their cause-effect has been debated. Here, we find that the saturated fat palmitate (PA) causes insulin resistance owing to failure of GLUT4 translocation in skeletal muscle myoblasts and myotubes without impairing signalling to Akt2 or AS160 (also known as TBC1D4). Instead, PA altered two basal-state events: (1) the intracellular localization of GLUT4 and its sorting towards a perinuclear storage compartment, and (2) actin filament stiffness, which prevents Rac1-dependent actin remodelling. These defects were triggered by distinct mechanisms, respectively protein palmitoylation and endoplasmic reticulum (ER) stress. Our findings highlight that saturated fats elicit muscle cell-autonomous dysregulation of the basal-state machinery required for GLUT4 translocation, which 'primes' cells for insulin resistance.

The resolution of phagosomes.

Phagocytosis is a fundamental immunobiological process responsible for the removal of harmful particulates. While the number of phagocytic events achieved by a single phagocyte can be remarkable, exceeding hundreds per day, the same phagocytic cells are relatively long-lived. It should therefore be obvious that phagocytic meals must be resolved in order to maintain the responsiveness of the phagocyte and to avoid storage defects. In this article, we discuss the mechanisms involved in the resolution process, including solute transport pathways and membrane traffic. We describe how products liberated in phagolysosomes support phagocyte metabolism and the immune response. We also speculate on mechanisms involved in the redistribution of phagosomal metabolites back to circulation. Finally, we highlight the pathologies owed to impaired phagosome resolution, which range from storage disorders to neurodegenerative diseases.

Junctional integrity and directional mobility of lymphatic endothelial cell monolayers are disrupted by saturated fatty acids.

The lymphatic circulation regulates transfer of tissue fluid and immune cells toward the venous circulation. While obesity impairs lymphatic vessel function, the contribution of lymphatic endothelial cells (LEC) to metabolic disease phenotypes is poorly understood. LEC of lymphatic microvessels are in direct contact with the interstitial fluid, whose composition changes during the development of obesity, markedly by increases in saturated fatty acids. Palmitate, the most prevalent saturated fatty acid in lymph and blood, is detrimental to metabolism and function of diverse tissues, but its impact on LEC function is relatively unknown. Here, palmitate (but not its unsaturated counterpart palmitoleate) destabilized adherens junctions in human microvascular LEC in culture, visualized as changes in VE-cadherin, α-catenin, and ß-catenin localization. Detachment of these proteins from cortical actin filaments was associated with abundant actomyosin stress fibers. The effects were Rho-associated protein kinase (ROCK)- and myosin-dependent, as inhibition with Y27632 or blebbistatin, respectively, prevented stress fiber accumulation and preserved junctions. Without functional junctions, palmitate-treated LEC failed to directionally migrate to close wounds in two dimensions and failed to form endothelial tubes in three dimensions. A reorganization of the lymphatic endothelial actin cytoskeleton may contribute to lymphatic dysfunction in obesity and could be considered as a therapeutic target.

Sex-related differences in patient characteristics, and efficacy and safety of advanced therapies in randomised clinical trials in psoriatic arthritis: a systematic literature review and meta-analysis.

BACKGROUND: Sex-related differences in clinical manifestations and disease outcomes exist in psoriatic arthritis, however, there is limited information on sex-related differences in randomised controlled trials of psoriatic arthritis. We aimed to compare patient characteristics and efficacy and safety of advanced therapies (including biological and targeted synthetic therapies) between male and female patients with psoriatic arthritis participating in randomised controlled trials. METHODS: In this systematic review and meta-analysis, we searched Medline, Embase, and Central databases, and conference abstract archives, from their inception to June 10, 2022, for randomised controlled trials that assessed the efficacy of advanced therapies in psoriatic arthritis. Two reviewers extracted information on participants' characteristics and rates of American College of Rheumatology (ACR) 20 and ACR50 response and minimal disease activity (MDA) by sex. Random-effects models were used to calculate pooled effects of ACR20, ACR50, and MDA in male versus female patients by drug class. FINDINGS: We included 54 trials (11 514 [50·9%] of 22 621 participants were female and 11 107 [49·1%] were male). Sex-disaggregated results were reported in a minority of studies (nine [17%] of 54 reported baseline characteristics by sex, 18 [33%] reported efficacy by sex, and two [4%] reported safety endpoints by sex). At baseline, male patients had lower baseline tender joint count (mean difference -3·01 [95% CI -3·83 to -2·18], health assessment questionnaire scores (-0·28 [-0·33 to -0·24]), pain scores (-4·58 [-6·86 to -2·30]), patient global assessment (-3·22 [-5·27 to -1·17]), and physician global assessment (-1·34 [-2·08 to -0·08]) than did female patients. Male patients had higher baseline psoriasis area and severity index scores (mean difference 1·95 [95% CI 0·78 to 3·11]) and C-reactive protein concentrations (2·57 [0·40 to 4·74]) than did female patients. ACR20 response by sex varied across drug classes, with higher rates in males than females with interleukin (IL)-17 inhibitors (odds ratio [OR] 1·70 [95% CI 1·38-2·11]), IL-23 inhibitor (1·46 [1·20-1·78]), IL-12 and IL-23 inhibitor (2·67 [1·39-5·09]), and tumour necrosis factor (TNF) inhibitors (1·55 [1·11-2·18]), but no difference with JAK and TYK2 inhibitors (1·10 [0·87-1·38]). Similarly, ACR50 response rates were higher in male patients versus female patients in all drug classes, with exception of JAK and TYK2 inhibitors (TNF inhibitors, OR 2·17 [95% CI 1·62-2·90]; IL-17 inhibitors, 1·93 [1·56-2·38]; IL-23 inhibitor, 1·71 [1·25-2·34]; IL-12 and 23 inhibitor, 2·43 [1·14-5·20]; and JAK and TYK2 inhibitors, 1·09 [0·73-1·62]). Male patients were more likely to reach MDA with most drug classes, including IL-17 inhibitors (OR 1·99 [95% CI 1·50-2·63]), IL-23 inhibitors (1·79 [1·29-2·50]), TNF inhibitors (2·62 [1·54-4·44]), and JAK and TYK2 inhibitors (1·77 [1·15-2·73]). Risk of bias was low for most studies. INTERPRETATION: Biological sex of patients with psoriatic arthritis influences their response to advanced therapies, but the effect varies by drug class. Selective reporting might have influenced these results. Future trials should report baseline characteristics and endpoint results by sex. FUNDING: Canadian Rheumatology Association.

The spectrin cytoskeleton integrates endothelial mechanoresponses.

Physiological blood flow induces the secretion of vasoactive compounds, notably nitric oxide, and promotes endothelial cell elongation and reorientation parallel to the direction of applied shear. How shear is sensed and relayed to intracellular effectors is incompletely understood. Here, we demonstrate that an apical spectrin network is essential to convey the force imposed by shear to endothelial mechanosensors. By anchoring CD44, spectrins modulate the cell surface density of hyaluronan and sense and translate shear into changes in plasma membrane tension. Spectrins also regulate the stability of apical caveolae, where the mechanosensitive PIEZO1 channels are thought to reside. Accordingly, shear-induced PIEZO1 activation and the associated calcium influx were absent in spectrin-deficient cells. As a result, cell realignment and flow-induced endothelial nitric oxide synthase stimulation were similarly dependent on spectrin. We conclude that the apical spectrin network is not only required for shear sensing but also transmits and distributes the resulting tensile forces to mechanosensors that elicit protective and vasoactive responses.

ARPC1B binds WASP to control actin polymerization and curtail tonic signaling in B cells.

Immune cells exhibit low-level, constitutive signaling at rest (tonic signaling). Such tonic signals are required for fundamental processes, including the survival of B lymphocytes, but when they are elevated by genetic or environmental causes, they can lead to autoimmunity. Events that control ongoing signal transduction are, therefore, tightly regulated by submembrane cytoskeletal polymers like F-actin. The actin-binding proteins that underpin the process, however, are poorly described. By investigating patients with ARPC1B deficiency, we report that ARPC1B-containing ARP2/3 complexes are stimulated by Wiskott Aldrich Syndrome protein (WASP) to nucleate the branched actin networks that control tonic signaling from the B cell receptor (BCR). Despite an upregulation of ARPC1A, ARPC1B-deficient cells were not capable of WASP-mediated nucleation by ARP2/3, and this caused the loss of WASP-dependent structures, including podosomes in macrophages and lamellipodia in B cells. In the B cell compartment, ARPC1B deficiency also led to weakening of the cortical F-actin cytoskeleton that normally curtails the diffusion of BCRs and ultimately resulted in increased tonic lipid signaling, oscillatory calcium release from the endoplasmic reticulum (ER), and phosphorylated Akt. These events contributed to skewing the threshold for B cell activation in response to microbial-associated molecular patterns (MAMPs). Thus, ARPC1B is critical for ARP2/3 complexes to control steady-state signaling of immune cells.

The cytoskeleton in phagocytosis and macropinocytosis.

Cells of the innate immune system, notably macrophages, neutrophils and dendritic cells, perform essential antimicrobial and homeostatic functions. These functions rely on the dynamic surveillance of the environment supported by the formation of elaborate membrane protrusions. Such protrusions - pseudopodia, lamellipodia and filopodia - facilitate the sampling of the surrounding fluid by macropinocytosis, as well as the engulfment of particulates by phagocytosis. Both processes entail extreme plasma membrane deformations that require the coordinated rearrangement of cytoskeletal polymers, which exert protrusive force and drive membrane coalescence and scission. The resulting vacuolar compartments undergo pronounced remodeling and ultimate resolution by mechanisms that also involve the cytoskeleton. Here, we describe the regulation and functions of cytoskeletal assembly and remodeling during macropinocytosis and phagocytosis.

C-terminus of Hsp70 Interacting Protein (CHIP) and Neurodegeneration: Lessons from the Bench and Bedside.

Neurodegenerative diseases are characterized by the increasing dysfunction and death of neurons, resulting in progressive impairment of a person's mobility and/or cognition. Protein misfolding and aggregation are commonly hypothesized to cause neurotoxicity and, eventually, neuronal degeneration that are associated with these diseases. Emerging experimental evidence, as well as recent findings from human studies, reveal that the C-terminus of Hsp70 Interacting Protein (CHIP), or STIP1 Homology and U-box containing Protein 1 (STUB1), is a quality control protein involved in neurodegeneration. Here, we review evidence that CHIP interacts with and plays a role in regulating proteins implicated in the pathogenesis of Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and polyglutamine diseases, including Huntington's disease and spinocerebellar ataxias. We also review clinical findings identifying mutations in STUB1 as a cause of both autosomal recessive and autosomal dominant forms of cerebellar ataxia. We propose that CHIP modulation may have therapeutic potential for the treatment of multiple neurodegenerative diseases.

Stabilization of Endothelial Receptor Arrays by a Polarized Spectrin Cytoskeleton Facilitates Rolling and Adhesion of Leukocytes.

Multivalent complexes of endothelial adhesion receptors (e.g., selectins) engage leukocytes to orchestrate their migration to inflamed tissues. Proper anchorage and sufficient density (clustering) of endothelial receptors are required for efficient leukocyte capture and rolling. We demonstrate that a polarized spectrin network dictates the stability of the endothelial cytoskeleton, which is attached to the apical membrane, at least in part, by the abundant transmembrane protein CD44. Single-particle tracking revealed that CD44 undergoes prolonged periods of immobilization as it tethers to the cytoskeleton. The CD44-spectrin "picket fence" alters the behavior of bystander molecules-notably, selectins-curtailing their mobility, inducing their apical accumulation, and favoring their clustering within caveolae. Accordingly, depletion of either spectrin or CD44 virtually eliminated leukocyte rolling and adhesion to the endothelium. Our results indicate that a unique spectrin-based apical cytoskeleton tethered to transmembrane pickets-notably, CD44-is essential for proper extravasation of leukocytes in response to inflammation.

Lipid-gated monovalent ion fluxes regulate endocytic traffic and support immune surveillance.

Despite ongoing (macro)pinocytosis of extracellular fluid, the volume of the endocytic pathway remains unchanged. To investigate the underlying mechanism, we used high-resolution video imaging to analyze the fate of macropinosomes formed by macrophages in vitro and in situ. Na+, the primary cationic osmolyte internalized, exited endocytic vacuoles via two-pore channels, accompanied by parallel efflux of Cl- and osmotically coupled water. The resulting shrinkage caused crenation of the membrane, which fostered recruitment of curvature-sensing proteins. These proteins stabilized tubules and promoted their elongation, driving vacuolar remodeling, receptor recycling, and resolution of the organelles. Failure to resolve internalized fluid impairs the tissue surveillance activity of resident macrophages. Thus, osmotically driven increases in the surface-to-volume ratio of endomembranes promote traffic between compartments and help to ensure tissue homeostasis.

Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling.

Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.

Contributions of the Hippocampal CA3 Circuitry to Acute Seizures and Hyperexcitability Responses in Mouse Models of Brain Ischemia.

The hippocampal circuitry is widely recognized as susceptible to ischemic injury and seizure generation. However, hippocampal contribution to acute non-convulsive seizures (NCS) in models involving middle cerebral artery occlusion (MCAO) remains to be determined. To address this, we occluded the middle cerebral artery in adult C57 black mice and monitored electroencephalographic (EEG) discharges from hippocampal and neocortical areas. Electrographic discharges in the absence of convulsive motor behaviors were observed within 90 min following occlusion of the middle cerebral artery. Hippocampal discharges were more robust than corresponding cortical discharges in all seizure events examined, and hippocampal discharges alone or with minimal cortical involvement were also observed in some seizure events. Seizure development was associated with ipsilateral hippocampal injuries as determined by subsequent histological examinations. We also introduced hypoxia-hypoglycemia episodes in mouse brain slices and examined regional hyperexcitable responses ex vivo. Extracellular recordings showed that the hippocampal CA3 region had a greater propensity for exhibiting single/multiunit activities or epileptiform field potentials following hypoxic-hypoglycemic (HH) episodes compared to the CA1, dentate gyrus, entorhinal cortical (EC) or neocortical regions. Whole-cell recordings revealed that CA3 pyramidal neurons exhibited excessive excitatory postsynaptic currents, attenuated inhibitory postsynaptic currents and intermittent or repetitive spikes in response to HH challenge. Together, these observations suggest that hippocampal discharges, possibly as a result of CA3 circuitry hyperexcitability, are a major component of acute NCS in a mouse model of MCAO.

Picket-fences in the plasma membrane: functions in immune cells and phagocytosis.

Recent studies of molecular mobility in the plasma membrane have revealed that diffusion is restricted by cytoskeletal networks or fences. Transmembrane protein "pickets" that reversibly associate with the membrane-associated skeleton and with the pericellular coat impede the movement of unattached bystander molecules. While membrane picket-fences were originally described as barriers to free diffusion in more passive cell types such as fibroblasts, they have particularly important functions in the more dynamic immune cells. In phagocytes, such fences curtail spontaneous activation and their disassembly facilitates stimulation by target particles, fostering receptor clustering and the exclusion of phosphatases from the phagocytic cup. In this review, we describe the nature of the cellular cytoskeleton and of the exoskeleton created by the pericellular coat, their association with transmembrane pickets, and the modulation of molecular mobility during phagocytosis.

Susceptibility to hippocampal kindling seizures is increased in aging C57 black mice.

The incidence of seizures increases with old age. Stroke, dementia and brain tumors are recognized risk factors for new-onset seizures in the aging populations and the incidence of these conditions also increased with age. Whether aging is associated with higher seizure susceptibility in the absence of the above pathologies remains unclear. We used classic kindling to explore this issue as the kindling model is highly reproducible and allows close monitoring of electrographic and motor seizure activities in individual animals. We kindled male young and aging mice (C57BL/6 strain, 2-3 and 18-22 months of age) via daily hippocampal CA3 stimulation and monitored seizure activity via video and electroencephalographic recordings. The aging mice needed fewer stimuli to evoke stage-5 motor seizures and exhibited longer hippocampal afterdischarges and more frequent hippocampal spikes relative to the young mice, but afterdischarge thresholds and cumulative afterdischarge durations to stage 5 motor seizures were not different between the two age groups. While hippocampal injury and structural alterations at cellular and micro-circuitry levels remain to be examined in the kindled mice, our present observations suggest that susceptibility to hippocampal CA3 kindling seizures is increased with aging in male C57 black mice.

Macrophages promote osteoblastic differentiation in-vivo: implications in fracture repair and bone homeostasis.

Macrophages are activated in inflammation and during early phases of repair processes. Interestingly, they are also present in bone during development, but their function during this process is unclear. Here, we explore the function of macrophages in bone development, growth, and repair using transgenic mice to constitutively or conditionally deplete macrophages. Depletion of macrophages led to early skeletal growth retardation and progressive osteoporosis. By 3 months of age, macrophage-deficient mice displayed a 25% reduction in bone mineral density and a 70% reduction in the number of trabecular bone compared to control littermates. Despite depletion of macrophages, functional osteoclasts were still present in bones, lining trabecular bone and the endosteal surface of the cortical bone. Furthermore, ablation of macrophages led to a 60% reduction in the number of bone marrow mesenchymal progenitor cells and a decrease in the ability of these cells to differentiate to osteoblasts. When macrophages were depleted during fracture repair, bone union was impaired. Calluses from macrophage-deficient animals were smaller, and contained less bone and more fibrotic tissue deposition. Taken together, this shows that macrophages are crucial for maintaining bone homeostasis and promoting fracture repair by enhancing the differentiation of mesenchymal progenitors.