Sustained activation of insulin receptors internalized in GLUT4 vesicles of insulin-stimulated skeletal muscle.

Exposure of target cells to insulin results in the formation of ligand receptor complexes on the cell surface and their subsequent internalization into the endosomal apparatus. A current view is that endocytosis of the insulin receptor (IR) kinase results in its rapid deactivation and sorting of the IR back to the cell surface or to late endocytic compartments. We report herein that, in skeletal muscle, in vivo stimulation with insulin induced a rapid internalization of the IR to an insulin-sensitive GLUT4-enriched intracellular membrane fraction. After 30 min of stimulation, IR content and tyrosine phosphorylation were increased by three and nine times in that fraction, respectively, compared with unstimulated muscles. In vitro autophosphorylation assays revealed that the kinase activity of internalized IRs was markedly augmented (eight to nine times) by insulin. In marked contrast with hepatic endosomes or adipocyte low-density microsomes, no IR tyrosine dephosphorylation activity was observed in GLUT4-enriched vesicles isolated from skeletal muscle. The activated IR was recovered in immunopurified GLUT4 vesicles after insulin stimulation. Insulin also increased tyrosine-phosphorylated insulin receptor substrate 1 and phosphatidylinositol 3-kinase adapter (p85) subunit contents in the intracellular membrane fraction, but these signaling molecules were not directly associated with GLUT4 vesicles. These results show that, in skeletal muscle, the activated IR reaches a GLUT4-enriched compartment where its activity is apparently sustained. We propose that compartmentalization of activated IRs to GLUT4 vesicles may play a role in sustaining insulin signaling at this locus in skeletal muscle.

The transferrin receptor defines two distinct contraction-responsive GLUT4 vesicle populations in skeletal muscle.

Insulin and contraction increase glucose transport in an additive fashion in skeletal muscle. However, it is still unclear whether they do so by inducing the recruitment of GLUT4 transporters from the same or distinct intracellular compartments to the plasma membrane and the T-tubules. Using the transferrin receptor as a recognized marker of recycling endosomes, we have examined whether insulin and/or contraction recruit GLUT4 from this pool to either the plasma membranes or T-tubules, isolated by subcellular fractionation of perfused hindlimb muscles. Either stimulus independently increased GLUT4 translocation from an intracellular fraction to both the plasma membrane and T-tubules. The combination of insulin and contraction induced a marked (approximately threefold) and almost fully additive increase in GLUT4 content, but only in the plasma membrane. Insulin did not stimulate transferrin receptor recruitment from the GLUT4-containing intracellular fraction to either the plasma membrane or the T-tubules. In contrast, contraction stimulated the recruitment of the transferrin receptor from the same GLUT4-containing intracellular fraction to the plasma membrane but not to the T-tubules. Contraction-induced recruitment of the transferrin receptor was also observed from immunopurified GLUT4 vesicles. It is concluded that muscle contraction stimulates translocation of GLUT4 from two distinct intracellular compartments: 1) a population of recycling endosomes that is selectively recruited to the plasma membrane and 2) from GLUT4 storage vesicles that are also insulin-responsive and recruited to both the plasma membrane and the T-tubules. The lack of additive translocation of GLUT4 to the T-tubules may be linked to the failure of GLUT4-containing recycling endosomes to be recruited to these structures.

Mechanism of impaired nitric oxide synthase activity in skeletal muscle of streptozotocin-induced diabetic rats.

AIMS/HYPOTHESIS: The aims of our study were to investigate whether nitric oxide synthase (NOS) activity is impaired in skeletal muscle of insulin-deficient [Type I (insulin-dependent)] diabetic rats and if the case, to determine the mechanism of NOS dysregulation in this disorder. METHODS: Rats were rendered diabetic by streptozotocin injection (65 mg/kg, i.v.) and NOS activity and expression in gastrocnemius muscles were studied 1, 2, 3 or 4 weeks after diabetes induction. RESULTS: The diabetic state was associated with a progressive reduction (down to 42 % of control values after 4 weeks) in muscle NOS activity compared with control rats. Using reverse transcriptase-polymerase chain reaction, we could not detect statistically significant changes in the expression of either neuronal NOS (nNOS) or endothelial NOS (eNOS) mRNAs in diabetic muscle. The contents of nNOS and eNOS protein were, however, progressively reduced in muscle homogenates of diabetic rats and these alterations were prevented by insulin treatment. Subcellular fractionation of skeletal muscle showed that both nNOS and eNOS proteins are mainly localised to the plasma membrane with lower abundance in T-tubules and not detectable in sarcoplasmic reticulum-enriched fractions. After 1 week of diabetes, eNOS protein content was decreased only in the plasma membrane whereas nNOS protein abundance was not affected at this time. Neither the expression nor the interaction of caveolin-1 and caveolin-3 with NOS enzymes was found to be altered in muscle of diabetic rats. CONCLUSION/INTERPRETATION: These results show that skeletal muscle NOS activity is impaired during the progression of insulin-deficient diabetes and reduced NOS activity is associated with a decreased abundance of both nNOS and eNOS proteins, which appears to involve post-transcriptional mechanisms.

Biochemical localisation of the 5-HT2A (serotonin) receptor in rat skeletal muscle.

The 5-HT2A receptor was recently shown to localise morphologically to the transverse tubules (TT) in rat foetal myoblasts. Receptor activation enhanced the expression of genes involved in myogenesis, and its TT localisation has led to the suggestion that it may participate in excitation-contraction coupling. In order to gain further insights into 5-HT2A receptor function in muscle we have (i) investigated its biochemical localisation in adult rat skeletal muscle and (ii) determined whether receptor expression is dependent upon muscle type. Immunoblot analysis of muscle membranes, isolated by subcellular fractionation, revealed that adult muscle expresses the 5-HT2A receptor and that it resides exclusively in plasma membranes and not in TT. No differences in 5-HT2A abundance were observed between red and white muscle, suggesting that receptor expression does not correlate with the metabolic or contractile properties of the muscle fibre. Our data indicate that 5-HT2A expression in skeletal muscle is maintained into adulthood and that its absence from TT make it an unlikely participant in the excitation-contraction coupling process.

Selective impairment in GLUT4 translocation to transverse tubules in skeletal muscle of streptozotocin-induced diabetic rats.

We previously reported that insulin induces the translocation of GLUT4 to both the plasma membrane and the transverse tubules (T-tubules) in rat skeletal muscle (Am J Physiol 270:E667-E676, 1996). The aim of the present study was to investigate whether the insulin-resistant glucose utilization of skeletal muscle from streptozotocin (STZ)-induced diabetic rats is linked to an impaired translocation of GLUT4 to the plasma membrane, the T-tubules, or both surface compartments. Whole-body insulin-mediated glucose disposal, assessed during a hyperinsulinemic-euglycemic clamp, was reduced by 48% (P < 0.01) in diabetic rats as compared with controls. Subcellular membrane fractions enriched with plasma membranes, T-tubules, or GLUT4-enriched intracellular membranes were isolated from hindlimb muscles of control and insulin-stimulated rats, and GLUT4 content was measured by Western blot analysis. In the absence of insulin (unstimulated), GLUT4 content in muscle of diabetic rats was markedly lower (by approximately 40%) in both the T-tubules and the intracellular membrane fraction as compared with controls. In contrast, the transporter protein levels were similar in the plasma membrane fraction. In skeletal muscle of control animals, the hyperinsulinemic clamp induced GLUT4 translocation from the intracellular membrane pool to both the plasma membrane and the T-tubule-enriched fractions (approximately 2.2-fold to approximately 2.5-fold). Surprisingly, insulin increased plasma membrane GLUT4 content to comparable levels in control and diabetic rat skeletal muscle. However, insulin-mediated GLUT4 translocation to the T-tubules was significantly reduced in the same muscle. Whole-body insulin action was significantly correlated with GLUT4 protein levels in the T-tubules, but not with the transporter content in either plasma membranes or intracellular membranes. These results strongly suggest that peripheral resistance to insulin action on glucose disposal in STZ-induced diabetic rats is caused by a selective impairment of GLUT4 translocation to skeletal muscle T-tubules.

Insulin-induced translocation of Na+-K+-ATPase subunits to the plasma membrane is muscle fiber type specific.

We have previously shown that an acute insulin treatment induces redistribution of the alpha 2- and beta 1- isoforms of the Na+-K+-ATPase from intracellular membranes to plasma membranes detected on subcellular fractionation of mixed muscles and immunoblotting with isoform-specific antibodies (H. S. Hundal et al. J. Biol. Chem. 267: 5040-5043, 1992). In the present study we give both biochemical and morphological evidence that this insulin effect is operative in muscles composed mostly of oxidative (red) fibers but not in muscles composed mostly of glycolytic (white) fibers. The redistribution of the Na+-K+-ATPase alpha 2- and beta 1-isoforms after insulin injection was detected in membranes isolated from and muscles (soleus, red gastrocnemius, red rectus femoris, and red vastus lateralis) but not in membranes from white muscles (white gastrocnemius, tensor fasciae latae, white rectus femoris, and white vastus lateralis). After insulin injection, the potassium-dependent 3-O-methylfluorescein phosphatase activity of the enzyme was higher by 22% in the plasma membrane-enriched fraction and lower by 15% in the internal membrane fraction isolated from red but not from white muscles. Quantitative immunoelectron microscopy of ultrathin muscle cryosections showed that in vivo insulin stimulation augmented the density of Na+-K+-ATPase alpha 2- and beta 1- isoforms at the plasma membrane of soleus muscle by 80 and 124%, respectively, with no change in white gastrocnemius muscle. The effect of insulin to increase the content of Na+-K+-ATPase alpha 2- and beta 1-subunits in isolated plasma membranes was still observed when glycemia was prevented from dropping by using hyperinsulinemic-euglycemic clamps. We conclude that the insulin-induced redistribution of the alpha 2- and beta 1-isoforms of the Na+-K+-ATPase from an intracellular pool to the plasma membrane in restricted to oxidative fiber-type skeletal muscles. This may be related to the selective expression of beta 1-subunits in these fibers and implies that the beta 2-subunit, typical of glycolytic muscles, does not sustain translocation of alpha 2 beta 2-complexes.

A new procedure for the isolation of plasma membranes, T tubules, and internal membranes from skeletal muscle.

A new subcellular fractionation procedure for the simultaneous isolation of plasma membranes and transverse (T) tubule membranes from a rat skeletal muscle was developed. This new technique allows the isolation and separation of plasma membranes and T tubules in distinct subcellular fractions, as revealed by the membrane distribution of enzymatic and immunologic markers of both cell surface compartments. The procedure also yields a novel membrane fraction that is devoid of markers of both surface domains but is markedly enriched with GLUT-4 glucose transporters, thus strongly suggesting that it represents an intracellular pool of GLUT-4. Using this new procedure, we found that acute in vivo insulin administration (30 min) increased GLUT-4 protein content in the plasma membrane and a T tubule fraction (by approximately 80%), whereas a smaller elevation (35%) was observed in another fraction enriched with T tubules. Insulin induced a concomitant reduction (approximately 40%) in GLUT-4 abundance in the intracellular fraction. These results further support the hypothesis that T tubules are involved in the regulation of glucose transport in skeletal muscle. This novel fractionation method will be useful in investigating the regulation of muscle GLUT-4 transporters in other physiological and disease states such as diabetes, where defective translocation of the transporter protein to either one or both cell surface domains is suspected to occur.

Marked depletion of GLUT4 glucose transporters in transverse tubules of skeletal muscle from streptozotocin-induced diabetic rats.

The principal goal of the present study was to determine the subcellular content of GLUT4 in diabetic rat muscle, and to test the hypothesis that a reduced abundance of the transporter protein in transverse tubules is responsible for impaired glucose utilization in that tissue. GLUT4 protein levels were measured in hindlimb muscle homogenates as well as in subcellular membrane fractions enriched with either plasma membranes, transverse tubules, or GLUT4-containing intracellular membranes from control and diabetic (streptozotocin-induced) rats. GLUT4 protein contents in diabetic muscle homogenates was reduced by 30% as compared to control rats. Subcellular fractionation experiments revealed that GLUT4 contents in transverse tubules-enriched fractions was markedly decreased (by 55-60%) in skeletal muscle of diabetic animals whereas no significant reductions in GLUT4 abundance was observed in the plasma membrane fraction. Moreover, GLUT4 was markedly depleted (by 45%) in the GLUT4-enriched intracellular membrane fraction. These results indicate that GLUT4 is markedly depleted in both the intracellular pool and in the cell surface membranes in muscle of STZ-diabetic rats. Most strikingly, this study demonstrates that transverse tubules and not the plasma membrane are the main sites of cell surface GLUT4 depletion in diabetic muscle.

Stability of milrinone in 0.45% sodium chloride, 0.9% sodium chloride, or 5% dextrose injections.

The stability of milrinone in 0.45% and 0.9% sodium chloride injections and in 5% dextrose injection in glass and plastic containers was studied. Admixtures containing milrinone 0.2 mg/mL were prepared in three 500-mL glass containers, three 500-mL polyethylpolypropyl copolymer plastic containers, and three 1-L flexible plastic containers of each solution. Milrinone content was determined by high-performance liquid chromatography at intervals during 72 hours of storage at room temperature; one sample of each solution and container type was protected from light. Duplicate assays of each sample were performed, and samples were observed for visual and pH changes. In all samples milrinone concentrations were more than 97% of the initial concentration. No changes in pH or appearance occurred. Milrinone at a concentration of 0.2 mg/mL is stable for 72 hours at room temperature in 0.45% and 0.9% sodium chloride injections and in 5% dextrose injection in glass or plastic containers.

Simplified automated procedure for measurement of certain catecholamine drugs delivered from aerosol inhalation units.

An automated colorimetric method is described for the measurement of certain catecholamine drugs, such as isoproterenol and epinephrine, in sample solutions derived from 2 metered doses delivered from the mouthpiece of aerosol inhalation units. The procedure, applicable to the expressed product, involves oxidizing the catecholamine with potassium ferricyanide at pH 6 to produce an aminochrome. This method is similar to the well known trihydroxyindole reaction procedure but differs in that the formed aminochrome is measured spectrophotometrically at 495 nm instead of being further derivatized by alkaline rearrangement to the trihydroxyindole species followed by either spectrophotometric or fluorometric measurement.

Fluoride uptake and dissolution behavior of a synthetic dental enamel-like substrate.

A new ceramic form of hydroxylapatite and enamel were found to behave similarly in regard to their acid dissolution behavior in the presence and absence of topically applied fluoride. Discrepancies between the two materials can be explained by morphological differences between the rough enamel and smooth ceramic particles.

Direct spectrophotometric determination of sulfathiazole in presence of sulfadiazine and sulfamerazine.

A direct spectrophotometric method for the quantitative determination of sulfathiazole and the total pyrimidyl sulfonamide content in a mixture containing sulfadiazine and sulfamerazine is reported. This three-component system was readily reduced to the problem of a simple two-component system analysis. Based on dual isoabsorptive wavelength spectroscopy, simultaneous equations were developed that required absorbance measurements at only two selected wavelengths both isoabsorptive. The location of the isoabsorptive wavelengths was dependent on the pH of the solvent medium, and 0.1 M HCl gave the best results. The validity of the derived equations was demonstrated in a recovery study involving synthetic mixtures containing varying quantities of the three sulfonamides. The recovery was linear over a wide concentration range, and the precision of the method was about 1%.

Determination of 17-monochloroacetylajmaline and its metabolite in plasma by TLC fluorescence detection.

A sensitive and specific method is described for the determination of 17-monochloroacetylajmaline (I) and its metabolite, ajmaline (II), in plasma. Method specificity is accomplished by combining an ion-pair extraction with chromatography followed by development and utilization of reaction product fluorescence of the isolated species on silica gel. Recovery of I and II added to plasma or water averaged 70%. The major loss in the assay resulted during a solvent evaporation step and was reproducible over the concentration interval studied. The limit of detectability for I is 0.06 mu-g/2 ml of plasma. The method was used to determine plasma levels of I and II in the dog following a dose of 10 mg/kg iv of I.