Autophagy in MHC class II antigen processing.

Durable adaptive immunity is dependent upon CD4 T-cell recognition of MHC class II molecules that display peptides from exogenous and endogenous antigens. Endogenously expressed cytosolic and nuclear antigens access MHC class II by way of several intracellular autophagic routes. These pathways include macroautophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy can deliver antigens into autophagosomes for processing by acidic proteases before MHC class II presentation. However, other endogenous antigens are processed by cytoplasmic proteases, yielding fragments that translocate via chaperone-mediated autophagy into the endosomal network to intersect MHC class II. Cross-talk between autophagy pathways, particularly in response to stress, appears to balance the relative efficiency of each pathway. This might limit redundancy, giving MHC class II broader access to antigens within intracellular compartments distinct from the endosomal network.

Safety, efficacy, and pharmacokinetic overview of low-dose daily administration of tadalafil.

INTRODUCTION: Several phosphodiesterase type 5 (PDE5) inhibitors are commercially available for the treatment of erectile dysfunction (ED). Development of the first once-daily alternative dosing regimen with a PDE5 inhibitor was motivated by the behavioral complexities associated with sexual intimacy. AIM: To provide an alternative dosing option for certain men who may benefit from the removal of the temporal linkage between administration of an ED therapy and sexual intimacy or for men and their partners who anticipate at least twice-weekly sexual activity. METHODS: Pharmacokinetic predictions of tadalafil plasma concentrations were generated based upon empirical data following 20-mg, single-dose administration coupled with tadalafil usage patterns from as-needed clinical trials. To support the pharmacokinetic simulations and pharmacodynamic assumptions, clinical trials were conducted to demonstrate the efficacy and safety of once-daily, low-dose tadalafil 2.5 and 5 mg. MAIN OUTCOME MEASURES: Simulated tadalafil plasma concentrations and comparison with safety and efficacy measures from clinical trials. RESULTS: Based upon pharmacodynamic and pharmacokinetic data, once-daily doses of tadalafil 5 mg were predicted to provide therapeutic concentrations that would be maintained throughout the 24-hour dosing interval. Additionally, for a subgroup of men who anticipate at least twice-weekly sexual activity and are currently taking tadalafil 20 mg, a reduction in daily tadalafil exposure was predicted. To support the hypothesis that low-dose, once-daily tadalafil may be a safe and effective treatment alternative, clinical trials were conducted to demonstrate the safety and efficacy of once-daily tadalafil 2.5 and 5 mg. These results were similar to those of historical as-needed studies evaluating tadalafil 10 and 20 mg. CONCLUSIONS: Consistent with pharmacokinetic predictions, data from clinical trials indicate that once-daily use of low-dose tadalafil is a safe and effective treatment for men with ED.

Chromium activates glucose transporter 4 trafficking and enhances insulin-stimulated glucose transport in 3T3-L1 adipocytes via a cholesterol-dependent mechanism.

Evidence suggests that chromium supplementation may alleviate symptoms associated with diabetes, such as high blood glucose and lipid abnormalities, yet a molecular mechanism remains unclear. Here, we report that trivalent chromium in the chloride (CrCl3) or picolinate (CrPic) salt forms mobilize the glucose transporter, GLUT4, to the plasma membrane in 3T3-L1 adipocytes. Concomitant with an increase in GLUT4 at the plasma membrane, insulin-stimulated glucose transport was enhanced by chromium treatment. In contrast, the chromium-mobilized pool of transporters was not active in the absence of insulin. Microscopic analysis of an exofacially Myc-tagged enhanced green fluorescent protein-GLUT4 construct revealed that the chromium-induced accumulation of GLUT4-containing vesicles occurred adjacent to the inner cell surface membrane. With insulin these transporters physically incorporated into the plasma membrane. Regulation of GLUT4 translocation by chromium did not involve known insulin signaling proteins such as the insulin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, and Akt. Consistent with a reported effect of chromium on increasing membrane fluidity, we found that chromium treatment decreased plasma membrane cholesterol. Interestingly, cholesterol add-back to the plasma membrane prevented the beneficial effect of chromium on both GLUT4 mobilization and insulin-stimulated glucose transport. Furthermore, chromium action was absent in methyl-beta-cyclodextrin-pretreated cells already displaying reduced plasma membrane cholesterol and increased GLUT4 translocation. Together, these data reveal a novel mechanism by which chromium may enhance GLUT4 trafficking and insulin-stimulated glucose transport. Moreover, these findings at the level of the cell are consistent with in vivo observations of improved glucose tolerance and decreased circulating cholesterol levels after chromium supplementation.

Phosphatidylinositol 4,5-bisphosphate reverses endothelin-1-induced insulin resistance via an actin-dependent mechanism.

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)) plays a pivotal role in insulin-stimulated glucose transport as an important precursor to PI 3,4,5-trisphosphate (PIP(3)) and a key regulator of actin polymerization. Since endothelin (ET)-1 impairs insulin sensitivity and PIP(2) is a target of ET-1-induced signaling, we tested whether a change in insulin-stimulated PIP(3) generation and signaling, PIP(2)-regulated actin polymerization, or a combination of both accounted for ET-1-induced insulin resistance. Concomitant with a time-dependent loss of insulin sensitivity, ET-1 caused a parallel reduction in plasma membrane PIP(2). Despite decreased insulin-stimulated PI 3-kinase activity and PIP(3) generation, ET-1 did not diminish downstream signaling to Akt-2. Furthermore, addition of exogenous PIP(2), but not PIP(3), restored insulin-regulated GLUT4 translocation and glucose transport impaired by ET-1. Microscopic and biochemical analyses revealed a PIP(2)-dependent loss of cortical filamentous actin (F-actin) in ET-1-treated cells. Restoration of insulin sensitivity by PIP(2) add-back occurred concomitant with a reestablishment of cortical F-actin. The corrective effect of exogenous PIP(2) in ET-1-induced insulin-resistant cells was not present in cells where cortical F-actin remained experimentally depolymerized. These data suggest that ET-1-induced insulin resistance results from reversible changes in PIP(2)-regulated actin polymerization and not PIP(2)-dependent signaling.

Endothelin-1 impairs glucose transporter trafficking via a membrane-based mechanism.

Endothelin-1 (ET-1) disrupts insulin-regulated glucose transporter GLUT4 trafficking. Since the negative consequence of chronic ET-1 exposure appears to be independent of signal disturbance along the insulin receptor substrate-1/phosphatidylinositol (PI) 3-kinase (PI3K)/Akt-2 pathway of insulin action, we tested if ET-1 altered GLUT4 regulation engaged by osmotic shock, a PI3K-independent stimulus that mimics insulin action. Regulation of GLUT4 by hyperosmotic stress was impaired by ET-1. Because of the mutual disruption of both insulin- and hyperosmolarity-stimulated GLUT4 translocation, we tested whether shared signaling and/or key phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated cytoskeletal events of GLUT4 trafficking were targets of ET-1. Both insulin and hyperosmotic stress signaling to Cbl were impaired by ET-1. Also, plasma membrane PIP2 and cortical actin levels were reduced in cells exposed to ET-1. Exogenous PIP2, but not PI 3,4,5-bisphosphate, restored actin structure, Cbl activation, and GLUT4 translocation. These data show that ET-1-induced PIP2/actin disruption impairs GLUT4 trafficking elicited by insulin and hyperosmolarity. In addition to showing for the first time the important role of PIP2-regulated cytoskeletal events in GLUT4 regulation by stimuli other than insulin, these studies reveal a novel function of PIP2/actin structure in signal transduction.

Interactions of endothelin and insulin: expanding parameters of insulin resistance.

Since the discovery of endothelin peptides in the mid-1980s by Yanigasawa and colleagues, accumulating evidence demonstrates that these peptides may function beyond vasoconstriction. Strong epidemiologic associations between insulin resistance and increased endothelin levels or activity have been found, and these associations have prompted studies investigating the interactions of endothelin with insulin. In this review we explore the evidence for such interactions at multiple levels of physiology, ranging from effects on tissue perfusion through modulation of vascular tone to subcellular interactions of endothelin signaling with insulin signaling. The evidence implicating endothelin in insulin resistance and its associated vascular and metabolic abnormalities is reviewed.

Disruption of cortical actin in skeletal muscle demonstrates an essential role of the cytoskeleton in glucose transporter 4 translocation in insulin-sensitive tissues.

Cell culture work suggests that signaling to polymerize cortical filamentous actin (F-actin) represents a required pathway for the optimal redistribution of the insulin-responsive glucose transporter, GLUT4, to the plasma membrane. Recent in vitro study further suggests that the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) mediates the effect of insulin on the actin filament network. Here we tested whether similar cytoskeletal mechanics are essential for insulin-regulated glucose transport in isolated rat epitrochlearis skeletal muscle. Microscopic analysis revealed that cortical F-actin is markedly diminished in muscle exposed to latrunculin B. Depolymerization of cortical F-actin with latrunculin B caused a time- and concentration-dependent decline in 2-deoxyglucose transport. The loss of cortical F-actin and glucose transport was paralleled by a decline in insulin-stimulated GLUT4 translocation, as assessed by photolabeling of cell surface GLUT4 with Bio-LC-ATB-BMPA. Although latrunculin B impaired insulin-stimulated GLUT4 translocation and glucose transport, activation of phosphatidylinositol 3-kinase and Akt by insulin was not rendered ineffective. In contrast, the ability of insulin to elicit the cortical F-actin localization of N-WASP was abrogated. These data provide the first evidence that actin cytoskeletal mechanics are an essential feature of the glucose transport process in intact skeletal muscle. Furthermore, these findings support a distal actin-based role for N-WASP in insulin action in vivo.

Protective effect of phosphatidylinositol 4,5-bisphosphate against cortical filamentous actin loss and insulin resistance induced by sustained exposure of 3T3-L1 adipocytes to insulin.

Muscle and fat cells develop insulin resistance when cultured under hyperinsulinemic conditions for sustained periods. Recent data indicate that early insulin signaling defects do not fully account for the loss of insulin action. Given that cortical filamentous actin (F-actin) represents an essential aspect of insulin regulated glucose transport, we tested to see whether cortical F-actin structure was compromised during chronic insulin treatment. The acute effect of insulin on GLUT4 translocation and glucose uptake was diminished in 3T3-L1 adipocytes exposed to a physiological level of insulin (5 nm) for 12 h. This insulin-induced loss of insulin responsiveness was apparent under both low (5.5 mm) and high (25 mm) glucose concentrations. Microscopic and biochemical analyses revealed that the hyperinsulinemic state caused a marked loss of cortical F-actin. Since recent data link phosphatidylinositol 4,5-bisphosphate (PIP(2)) to actin cytoskeletal mechanics, we tested to see whether the insulin-resistant condition affected PIP(2) and found a noticeable loss of this lipid from the plasma membrane. Using a PIP(2) delivery system, we replenished plasma membrane PIP(2) in cells following the sustained insulin treatment and observed a restoration in cortical F-actin and insulin responsiveness. These data reveal a novel molecular aspect of insulin-induced insulin resistance involving defects in PIP(2)/actin regulation.

Sphingomyelinase activates GLUT4 translocation via a cholesterol-dependent mechanism.

A basis for the insulin mimetic effect of sphingomyelinase on glucose transporter isoform GLUT4 translocation remains unclear. Because sphingomyelin serves as a major determinant of plasma membrane cholesterol and a relationship between plasma membrane cholesterol and GLUT4 levels has recently become apparent, we assessed whether GLUT4 translocation induced by sphingomyelinase resulted from changes in membrane cholesterol content. Exposure of 3T3-L1 adipocytes to sphingomyelinase resulted in a time-dependent loss of sphingomyelin from the plasma membrane and a concomitant time-dependent accumulation of plasma membrane GLUT4. Degradation products of sphingomyelin did not mimic this stimulatory action. Plasma membrane cholesterol amount was diminished in cells exposed to sphingomyelinase. Restoration of membrane cholesterol blocked the stimulatory effect of sphingomyelinase. Increasing concentrations of methyl-beta-cyclodextrin, which resulted in a dose-dependent reversible decrease in membrane cholesterol, led to a dose-dependent reversible increase in GLUT4 incorporation into the plasma membrane. Although increased plasma membrane GLUT4 content by cholesterol extraction with concentrations of methyl-beta-cyclodextrin above 5 mM most likely reflected decreased GLUT4 endocytosis, translocation stimulated by sphingomyelinase or concentrations of methyl-beta-cyclodextrin below 2.5 mM occurred without any visible changes in the endocytic retrieval of GLUT4. Furthermore, moderate loss of cholesterol induced by sphingomyelinase or low concentrations of methyl-beta-cyclodextrin did not alter membrane integrity or increase the abundance of other plasma membrane proteins such as the GLUT1 glucose transporter or the transferrin receptor. Regulation of GLUT4 translocation by moderate cholesterol loss did not involve known insulin-signaling proteins. These data reveal that sphingomyelinase enhances GLUT4 exocytosis via a novel cholesterol-dependent mechanism.