Transient changes in four GLUT4 compartments in rat adipocytes during the transition, insulin-stimulated to basal: implications for the GLUT4 trafficking pathway.

In rat adipocytes, insulin-induced GLUT4 recruitment to the plasma membrane (PM) is associated with characteristic changes in the GLUT4 contents of three distinct endosomal fractions, T, H, and L. The organelle-specific marker distribution pattern suggests that these endosomal GLUT4 compartments are sorting endosomes (SR), GLUT4-storage endosomes (ST), and GLUT4 exocytotic vesicules (EV), respectively, prompting us to analyze GLUT4 recycling based upon a four-compartment kinetic model. Our analysis revealed that insulin modulates GLUT4 trafficking at multiple steps, including not only the endocytotic and exocytotic rates, but also the two rate coefficients coupling the three intracellular compartments. This analysis assumes that GLUT4 cycles through PM T, H,L, and back to PM, in that order, with transitions characterized by four first-order coefficients. Values assigned to these coefficients are based upon the four steady-state GLUT4 pool sizes assessed under both basal and insulin stimulated states and the transition time courses observed in the plasma membrane GLUT4 pool. Here we present the first reported experimental measurements of transient changes in each of the four GLUT4 compartments during the insulin-stimulated to basal transition in rat adipocytes and compare these experimental results with the corresponding model simulations. The close correlation of these results offers clear support for the general validity of the assumed model structure and the assignment of the T compartment to the sorting endosome GLUT4 pool. Variations in the recycling pathway from that of an unbranched cyclic topography are also considered in the light of these experimental observations. The possibility that H is a coupled GLUT4 storage compartment lying outside the direct cyclic pathway is contraindicated by the data. Okadaic acid-induced GLUT4 recruitment is accompanied by modulation of the rate coefficients linking individual endosomal GLUT4 compartments, further demonstrating a significant role of the endosomal pathways in GLUT4 exocytosis.

Zinc tetrakis(N-methyl-4'-pyridyl) porphyrinato is an effective inhibitor of stimulant-induced activation of RAW 264.7 cells.

One proposed mechanism for the development of silica-induced fibrosis is prolonged pulmonary inflammation and lung damage resulting from the secretion of reactive mediators from alveolar macrophages. Metalloporphyrins have antioxidative and antiinflammatory activities. However, the molecular basis for the antiinflammatory action of zinc tetrakis(N-methyl-4'-pyridyl) porphyrinato (ZnTMPyP) has not been elucidated. The objective of this study was to determine whether ZnTMPyP exhibited the ability to inhibit the production of reactive oxygen species (ROS), the activation of NF-kappaB, or the secretion of IL-1 in RAW 264.7 cells, and whether such inhibitory activity was related to the ROS-scavenging ability of ZnTMPyP. The results indicate that, although ZnTMPyP is not cytotoxic to RAW 264.7 cells, it is a potent inhibitor in ROS production by RAW 264.7 cells in response to various stimulants, such as silica, zymosan, or phorbol myristate acetate. ZnTMPyP is also effective in reducing stimulant-induced DNA-binding activity of NF-kappaB and silica-induced tyrosine phosphorylation of IkappaB-alpha. ZnTMPyP also inhibits LPS-induced IL-1 production. However, ZnTMPyP exhibits relatively weak ability to directly scavenge hyroxyl or superoxide radicals. On the basis of effective concentrations of ZnTMPyP, these results suggest that ZnTMPyP directly acts as an inhibitor of cellular activation in addition to exhibiting an antioxidant effect. Therefore, it is suggested that further studies concerning the effects of ZnTMPyP using in vivo oxidative stress models or its effects on the cytotoxic process of human diseases associated with lung inflammation and injury are warranted. In addition, ZnTMPyP may be a useful tool to investigate the molecular mechanisms involved in stimulant-induced signal pathways.

Lateral plantar neuropathy.

We report 8 cases of lateral plantar neuropathy (LPN). All had sensory impairment over the territory of the lateral plantar nerve. Near-nerve needle sensory nerve conduction study (NCS) of the plantar nerves showed abnormality confined to the lateral plantar nerve, confirming LPN. The most common cause for LPN was trauma and the most common site of injury was at the passage of the lateral plantar nerve through the abductor tunnel at the instep of the foot.

The accessory nerve repetitive nerve stimulation test: a valuable second-line test in myasthenia gravis.

The diagnostic usefulness of the accessory nerve repetitive nerve stimulation (RNS) test was evaluated in 100 patients with myasthenia gravis (MG). The test was easy to perform and reliable at the low rates of stimulation. A higher diagnostic sensitivity was found in the accessory nerve RNS test than in the ulnar nerve RNS test on either the abductor digiti quinti or flexor carpi ulnaris muscles, especially in mild generalized MG. Diagnostic sensitivity was significantly increased when RNS test results for three muscles were combined, especially in mild generalized MG and sero-positive MG. In a small number of cases only the ulnar or accessory nerve RNS test was abnormal. There was a good correlation between electrophysiological and clinical severity of MG in the accessory nerve RNS test Thus, we conclude that the accessory nerve RNS test is a valuable second-line test and its greatest usefulness is in cases of mild generalized MG.

Transport function and subcellular distribution of purified human erythrocyte glucose transporter reconstituted into rat adipocytes.

In order to delineate the insulin-independent (constitutive) and insulin-dependent regulations of the plasma membrane glucose transporter concentrations in rat adipocytes, we introduced purified human erythrocyte GLUT-1 (HEGT) into rat adipocytes by poly(ethylene glycol)-induced vesicle-cell fusion and its transport function and subcellular distribution in the host cell were measured. HEGT in adipocytes catalysed 3-O-methylglucose equilibrium exchange with a turnover number that is indistinguishable from that of the basal adipocyte transporters. However, insulin did not stimulate significantly the HEGT function in adipocytes where it stimulated the native transporter function by 7-8-fold. The steady state distribution and the transmembrane orientation assays revealed that more than 85% of the HEGT that were inserted in the physiological, cytoplasmic side-in orientation at the adipocytes plasma membrane were moved into low-density microsomes (LDM), while 90% of the HEGT that were inserted in the wrong, cytoplasmic side-out orientation were retained in the plasma membrane. Furthermore, more than 70% of the LDM-associated HEGT were found in a small subset of LDM that also contained 80% of the LDM-associated GLUT-4, the insulin-regulatable, native adipocyte glucose transporter. However, insulin did not cause redistribution of HEGT from LDM to the plasma membrane under the condition where it recruited GLUT-4 from LDM to increase the plasma membrane GLUT-4 content 4-5-fold. These results demonstrate that the erythrocyte GLUT-1 introduced in adipocytes transports glucose with an intrinsic activity similar to that of the adipocyte GLUT-1 and/or GLUT-4, and enters the constitutive GLUT-4 translocation pathway of the host cell provided it is in physiological transmembrane orientation, but fails to enter the insulin-dependent GLUT-4 recruitment pathway. We suggested that the adipocyte plasma membrane glucose transporter concentration is constitutively kept low by a mechanism where a cell-specific constituent interacts with a cytoplasmic domain common to GLUT-1 and GLUT-4, while the insulin-dependent recruitment requires a cytoplasmic domain specific to GLUT-4.

Phenylarsine oxide causes an insulin-dependent, GLUT4-specific degradation in rat adipocytes.

An incubation of rat adipocytes with phenylarsine oxide (PAO) and then with insulin caused an inhibition of 3-O-methylglucose equilibrium exchange flux and a parallel reduction in cellular GLUT4 content detected by Western blots. Both the transport inhibition and the GLUT4 reduction were saturable with an increasing concentration of PAO showing essentially an identical Ki value of 35 microM. Both effects were not observed in the absence of insulin or if cells were incubated with insulin first. The reduction was specific to GLUT4; the immunoreactivities of GLUT1, insulin receptor, and clathrin were not affected in these experiments. The GLUT4 reduction occurred only in intact cells and was not observed in homogenized cells or fractionated membranes. GLUT4 in both the microsomal storage pool and the plasma membrane pool were affected with no indication of insulin-induced recruitment impairment. GLUT4 reduction was not observed in the presence of chloroquine or at 18 degrees C suggesting involvement of the lysosomal pathway. Based on these results, we propose that there is a PAO-sensitive protein mechanism that controls an insulin-dependent GLUT4 degradation pathway in adipocytes. This protein mechanism and the GLUT4 degradation pathway may play an important role in determining the steady-state GLUT4 level in the insulin-sensitive peripheral tissues in normal and diseased states.

Aggregation and fusion of unilamellar vesicles by poly(ethylene glycol).

Various aspects of the interaction between the fusogen, poly(ethylene glycol) and phospholipids were examined. The aggregation and fusion of small unilamellar vesicles of egg phosphatidylcholine (PC), bovine brain phosphatidylserine (PS) and dimyristoylphosphatidylcholine (DMPC) were studied by dynamic light scattering, electron microscopy and NMR. The fusion efficiency of Dextran, glycerol, sucrose and poly(ethylene glycol) of different molecular weights were compared. Lower molecular weight poly(ethylene glycol) are less efficient with respect to both aggregation and fusion. The purity of poly(ethylene glycol) does not affect its fusion efficiency. Dehydrating agents, such as Dextran, glycerol and sucrose, do not induce fusion. 31P-NMR results revealed a restriction in the phospholipid motion by poly(ethylene glycol) greater than that by glycerol and Dextran of similar viscosity and dehydrating capacity. This may be associated with the binding of poly(ethylene glycol) to egg PC, with a binding capacity of 1 mol of poly(ethylene glycol) to 12 mol of lipid. Fusion is greatly enhanced below the phase transition for DMPC, with extensive fusion occurring below 6% poly(ethylene glycol). Fusion of PS small unilamellar vesicles depends critically on the presence of cations. Large unilamellar vesicles were found to fuse less readily than small unilamellar vesicles. The results suggest that defects in the bilayer plays an important role in membrane fusion, and the 'rigidization' of the phospholipid molecules facilitates fusion possibly through the creation of defects along domain boundaries. Vesicle aggregation caused by dehydration and surface charge neutralization is a necessary but not a sufficient condition for fusion.

Effects of physical states of phospholipids on the incorporation and cytochalasin B binding activity of human erythrocyte membrane proteins in reconstituted vesicles.

Proteoliposomes were reconstituted from a Triton extract of human erythrocyte membrane proteins and a mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of varying ratios. With mixtures of egg PC and soybean PE, the protein/lipid ratio of the reconstituted vesicles was maximal at 25% PC and 75% PE, the composition which is known to have a maximum bilayer disruption (highest occurrence of lipidic particles seen by freeze-fracture electron microscopy). With mixtures of 1-palmitoyl-2-oleoyl-PC and dilinoleoyl-PE, which gave vesicles with few isolated lipidic particles at room temperature, the effect was less pronounced. The specific activity of the cytochalasin B (CB) binding protein in the reconstituted vesicles, on the other hand, was increased monotonically up to severalfold as the PC content was increased in the egg PC/soybean PE mixture. A similar increase was observed when soybean PE was partially substituted by dimyristoyl-PC, cholesterol, or transphosphatidylated PE from egg PC. These findings indicate that preexisting defects in the lipid bilayer promote protein incorporation into the bilayer during reconstitution whereas reduction of the bilayer fluidity facilitates the CB binding activity in the reconstituted vesicles.

Glucose transport carrier of human erythrocytes. Radiation-target size of glucose-sensitive cytochalasin B binding protein.

Apparent molecular sizes of D-glucose-sensitive cytochalasin B binding protein of human erythrocyte membranes are assessed by applying classical target theory to irradiation-inactivation data. Molecular weights of this protein as it occurs in untreated ghosts, EDTA-treated ghosts, and reconstituted vesicles of Triton extract of ghosts are 220,000, 180,000, and 220,000, respectively. These results, in conjunction with other findings in the literature, suggest that the native form of the glucose transport carrier of human erythrocytes is a tetrameric assembly of a 50,000-dalton monomer or is a dimer of 100,000 daltons.