Effects of fluorescein isothiocyanate on insulin actions in rat adipocytes.

The effects of fluorescein isothiocyanate II (FITC) on the actions of insulin in rat adipocytes were studied. When adipocytes were incubated with FITC at pH 7.4 (2 mM agent, 8 min), the cells were completely deprived of their specific insulin-binding activity and rendered unresponsive to the hormone. The effect of FITC on the insulin-binding activity was milder at pH 9.0, and cAMP phosphodiesterase in cells exposed to FITC at pH 9.0 was maximally stimulated if the insulin concentration was increased to 100 nM. Under identical conditions, however, glucose transport activity was rendered not only less sensitive but also less responsive to the hormone. When FITC was added to cells after insulin at pH 9.0, the glucose transport activity that had been stimulated by the hormone was considerably reduced. This reduction was largely, but not entirely, prevented if the cells were deprived of ATP, suggesting that FITC (a) elicited the ATP-dependent reversal of the hormonal effect and, simultaneously, (b) mildly inhibited the transport activity per se. Western blot assay of GLUT-4 (a major isoform of glucose transporter in adipocytes) indicated that FITC (a) partially blocked insulin-dependent translocation of GLUT-4 from the intracellular site to the plasma membrane while it (b) induced a mild "insulin-like" effect. It is concluded that FITC at pH 9.0 (a) renders both glucose transport and phosphodiesterase activities less insulin sensitive presumably by modifying the cellular hormone receptor and (b) makes glucose transport activity less responsive to insulin presumably by (i) blocking hormone-dependent translocation of glucose transporter and (ii) mildly inhibiting intrinsic glucose transport activity.

Cyclic GMP-dependent stimulation of the membrane-bound insulin-sensitive cAMP phosphodiesterase from rat adipocytes.

The insulin-sensitive cAMP phosphodiesterase (phosphodiesterase) in rat adipocytes is a membrane-bound low Km enzyme that can be recovered in a crude microsomal fraction (Fraction P-2). The action of this enzyme to hydrolyze cAMP is known to be inhibited by cGMP; nevertheless, it was found in our present study that under selected conditions, the enzyme can also be stimulated by cGMP as well as some other nucleotide derivatives. The maximum cGMP-dependent stimulation was observed when the enzyme in Fraction P-2 was incubated with 10 microM cGMP for 5-20 min at 37 degrees C in the presence of Mg2+, washed, and then assayed in the absence of added cGMP. The level of this stimulation was close to, but less than, that achieved by insulin in intact cells. The actions of the cGMP- and insulin-stimulated enzymes to hydrolyze labeled cAMP were inhibited in an identical manner by cilostamide (Ki = 0.10 microM), griseolic acid (Ki = 0.19 microM), unlabeled cAMP (Km = 0.20 microM), and cGMP (Ki = 0.16 microM), all added to the assay system. Also, the basal, insulin-stimulated, and cGMP-activated enzymes were identically inhibited by a polyclonal antibody raised against a purified membrane-bound low Km phosphodiesterase from bovine adipose tissue. When the same antibody was used for the Western blot analysis of Fraction P-2, it immunoreacted with a single band of protein (165 kDa). These observations indicate that the insulin-sensitive phosphodiesterase in rat adipocytes can be stimulated with 10 microM cGMP and that this stimulation is detectable only after the nucleotide has been eliminated since the enzyme would be strongly inhibited by the nucleotide if the latter exists in the assay system. It is proposed that the insulin-sensitive phosphodiesterase, which is often referred to as a Type IV enzyme, is functionally similar to the Type II enzymes that are known to be stimulated by a low concentration of cGMP and inhibited by higher concentrations of the same nucleotide.

Diversity in the effects of extracellular ATP and adenosine on the cellular processing and physiologic actions of insulin in rat adipocytes.

ATP or adenosine (1 mM) added to extracellular buffer abolished both chloroquine- and monensin-dependent accumulation of [125I]iodoinsulin in isolated rat adipocytes. The effects of ATP were not secondary to its conversion to adenosine and were mimicked by beta, gamma-methyleneadenosine 5'-triphosphate. ATP, but not adenosine, partially inhibited the binding of insulin to the cellular receptor. Neither ATP nor adenosine had any significant effect on both internalization of cell-bound insulin and externalization of the internalized hormone. The degradation of cell-bound insulin was reduced to a considerable extent by both 0.1 mM chloroquine and 5 mM ATP, to a lesser degree by 1 mM ATP, and not significantly by 1 or 5 mM adenosine. Physiologically, (a) 1 mM ATP had a strong, while 1 mM adenosine had a mild inhibitory effect on the insulin-stimulated glucose transport without affecting its basal activity, (b) both ATP and adenosine moderately stimulated basal as well as insulin-stimulated glycogen synthase, and (c) ATP, but not adenosine, transiently stimulated basal cAMP phosphodiesterase without affecting the insulin-stimulated enzyme. Phosphodiesterase in cells that had been exposed to ATP for 30 min was refractory to ATP added afresh, but not to insulin. These data suggest that (a) extracellular ATP may block the degradative pathway of insulin processing, (b) adenosine might render the ordinarily irreversible intracellular traffic of insulin reversible or modulate a pathway which is yet to be identified, (c) the previously reported effect of ATP on glycogen synthase may not involve phosphorylation, (d) ATP stimulates cAMP phosphodiesterase by a mechanism which is distinct from that of insulin, and (e) the degradative pathway of insulin processing may not be involved in the physiologic actions of the hormone on glycogen synthase and phosphodiesterase.

Sedimentation characteristics of vesicles associated with insulin-sensitive intracellular glucose transporter from rat adipocytes.

The sedimentation characteristics of vesicles associated with the insulin-sensitive intracellular glucose transporter from rat adipocytes were studied. The method used was sucrose density gradient centrifugation, which was carried out under non-equilibrium and equilibrium (isopycnic) conditions. The glucose transport activity was determined by the reconstitution method. As reported previously, the sedimentation velocity of the intracellular glucose-transport activity was considerably slower than that of the counterpart in the plasma membrane. It was found, however, that the specific gravity of the slow-sedimenting glucose-transport activity was almost identical to that of the activity in the plasma membrane (d = 1.118-1.122). It is concluded that the intracellular glucose transport activity is associated not with low-density microsomal vesicles, but with unidentified slow-sedimenting vesicles that have a specific gravity similar to that of the plasma membrane.

Reassessment of insulin effects on the Vmax and Km values of hexose transport in isolated rat epididymal adipocytes.

Effects of insulin on the kinetic parameters of hexose transport in rat epididymal adipocytes were re-examined. The transport activity was assessed by measuring the rate of uptake of 3-O-[3H]methyl-D-glucose (MeGlc) under equilibrium exchange and zero-trans conditions. The incubation was carried out at 37 degrees C in an infant incubator. During the incubation, the cell suspension (25%, v/v, in a total volume of 48 microliter) was mechanically swirled at a rate of 600 rpm (r = 2 mm). The swirling facilitated the rapid uptake of MeGlc without stimulating the basal transport activity by "mechanical agitation". The basal and insulin-treated cells were incubated under identical conditions, except for the length of the incubation period. The incubation was terminated by the addition of 350 microliters of 1 mM phloretin, which inhibited transport in approximately 0.06 s. The time course of MeGlc uptake was consistent with the view that the process was a multiple-phase reaction. The initial phase of the reaction was completed when the intracellular distribution space of MeGlc was approximately 1% of the total cell volume. Insulin (10 nM) increased the Vmax value of MeGlc uptake 16-fold in equilibrium exchange experiments and 18-fold in zero-trans experiments. At the same time, the hormone decreased the Km value of MeGlc uptake from 11.7 to 5.4 mM in equilibrium exchange experiments and from 9.7 to 4.8 mM in zero-trans experiments. It is concluded that the major effect of insulin on MeGlc uptake is to increase the Vmax value, but the hormone has the additional effect of lowering the apparent Km value.

Apparent translocation of glucose transport activity in rat epididymal adipocytes by insulin-like effects of high pH or hyperosmolarity.

The basal and plus insulin states of glucose transport activity in adipocytes are known to show different responses to changes in the pH or osmolarity of the incubation mixture. When the pH was raised from 7 to 8, the basal glucose transport activity (assessed from the rate of 3-O-methyl-D-glucose uptake) was increased approximately 3-fold while the plus insulin activity remained virtually unaffected. Likewise, when cells were exposed to 300 mM sorbitol, the basal glucose transport activity, but not the plus insulin activity, was considerably increased. In both cases, the change in the transport activity was ATP-dependent and was completed in approximately 60 min. The increase in the cellular glucose transport activity was accompanied, in both cases, by an increase in the glucose transport activity in the plasma membrane fraction and a decrease in the activity in the high-speed pellet fraction. The transport activity in the subcellular fractions was determined after reconstitution into egg lecithin liposomes. Both isotonic buffer at pH 8.0 and hypertonic buffer at pH 7.4 significantly stimulated membrane-bound cAMP phosphodiesterase in adipocytes. It is concluded that the above two experimental conditions may induce insulin-like effects in fat cells and may facilitate translocation of the glucose transport activity from an intracellular site to the plasma membrane.