Receptor-mediated degradation of insulin in isolated rat adipocytes. Formation of a degradation product slightly smaller than insulin

More than 90% of the radioactivity associated with isolated rat adipocytes incubated with [TyrA14-125I]monoiodoinsulin represented at steady state iodoinsulin possessing full binding affinity. In contrast, about half of the radioactivity dissociating from the cells was [125I]monoiodotyrosine. The other half was of a molecular size similar to that of iodoinsulin as judged from gel-filtration chromatography. However, the descending limb of the 'insulin' peak (i.e., the smaller molecules) possessed a reduced binding activity compared with native iodoinsulin, material from the ascending limb, or a similar fraction isolated from dissociation medium from IM-9 lymphocytes, a cell type devoid of receptor-mediated insulin degradation. The cells, thus, release an intermediary degradation product.     

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.     

Receptor-mediated degradation of insulin in isolated rat adipocytes: Formation of a degradation product slightly smaller than insulin

More than 90% of the radioactivity associated with isolated rat adipocytes incubated with [Tyr^A14-¹²⁵I]monoiodoinsulin represented at steady state iodoinsulin possessing full binding affinity. In contrast, about half of the radioactivity dissociating from the cells was [¹²⁵I]monoiodotyrosine. The other half was of a molecular size similar to that of iodoinsulin as judged from gel-filtration chromatography. However, the descending limb of the ‘insulin’ peak (i.e., the smaller molecules) possessed a reduced binding activity compared with native iodoinsulin, material from the ascending limb, or a similar fraction isolated from dissociation medium from IM-9 lymphocytes, a cell type devoid of receptor-mediated insulin degradation. The cells, thus, release an intermediary degradation product.     

Reversal of insulin effects in fat cells may require energy for deactivation of glucose transport, but not deactivation of phosphodiesterase

The reversal of insulin effects on sugar transport and phosphodiesterase in fat cells was studied after arresting further actions of insulin with KCN, NaN₃, 2,4-dinitrophenol, or dicumarol. These agents rapidly lower the ATP concentration and concomitantly block the actions of insulin added later. Contrary to our expectation, the above inhibitors failed to initiate deactivation of the hormone-stimulated transport system. Instead, in the presence of the agents the transport system remained activated even after cells had been washed with an insulin-free buffer. This effect of the inhibitors was reversed when cells were washed with an inhibitor-free buffer containing glucose or pyruvate. The above inhibitors also blocked the deactivation of sugar transport stimulated by mechanical agitation. The effects of the inhibitors could not be explained by their possible effects on the basal transport activity, the intracellular urea space, or the cell count. The insulin-stimulated phosphodiesterase activity was rapidly lowered when cells were exposed to the above inhibitors. Apparently, these agents did not denature phosphodiesterase itself since the latter could be reactivated by insulin when inhibitor-treated cells were washed with a glucose-containing buffer. None of the above agents, except dicumarol, significantly inhibited phosphodiesterase activity in a cell-free system. It is suggested that the effects of insulin on sugar transport and phosphodiesterase are reversed by different mechanisms. ATP or metabolic energy may be involved in the deactivation of sugar transport, but not in that of phosphodiesterase.     

Binding, internalization, and lysosomal association of 125I-human growth hormone in cultured human lymphocytes: a quantitative morphological and biochemical study

125I-human growth hormone (125I-hGH) binds specifically to receptors on cultures human lymphocytes (IM-9). When this process is studied by use of quantitative EM radioautography, under conditions of incubation at 15 degrees C for 5 min, the ligand is localized to the plasma membrane of the cell. At 30 degrees and 37 degrees C, however, 125I-hGH is progressively internalized by the cell as a function of time. The internalized ligand is found predominantly in the Golgi region of the cells, with a five-fold preferential localization to membrane-bounded structures with the morphological and cytochemical characteristics of lysosomes. Up to 59% of these lysosome-like structures are positive for the acid phosphatase reaction under the conditions of incubation at 37 degrees C for 120 min. When the cell associated radioactivity after 15- 120 min of incubation at 37 degrees C is extracted in 1 M acetic acid and filtered on a Sephadex G-100 column, 58-73% of the material elutes as intact hGH. When cells are incubated with 125I-hGH at 37 degrees C for 15-120 min, separated from the incubation medium, and washed and diluted 100-fold, the percent 125I-hGH dissociable decreases as a function of increasing time of incubation. When cells are incubated with 125I-hGH for 15 min at 37 degrees C and the radioactivity that dissociates from the cells during 15-90 min is studied, the labeled material appearing in the incubation medium is progressively degraded as a function of time of incubation. When the dissociation process is studied radioautographically, grains are found both in plasma membrane and intracelluar compartments after 30 min of association, but after 30 and 120 min of dissociation a higher proportion of grains are in the intracellular compartment. After 120 min of association, there is less dissociation from either compartment and a preferential increase of grains in the intracellular compartment. These data suggest that receptor-linked internalization of a polypeptide hormone provides a mechanism that couples degradation of the ligand with loss of the cell surface receptor.     

Insulin processing and signal transduction in rat adipocytes

A glycine-HCl buffer (glycine, 50 mM/NaCl, 0.15 M/HCl, pH 3.5) was used to strip insulin bound to adipocyte cell surfaces. Adipocytes retained their integrity in the glycine buffer and their binding capacity for [125I]iodoinsulin could be completely recovered on transfer of the cells to physiological media. At 37 degrees C, [125I]iodoinsulin binds rapidly to plasma membrane receptors; maximal binding occurs within 10 min. At this temperature, the initial binding is followed by rapid internalization, degradation of the hormone and subsequent loss of label. Insulin treatment, at 37 degrees C, induced internalization of 37% of the plasma membrane insulin receptors. Phenylarsine oxide (PAO), a confirmed inhibitor of protein internalization, allowed insulin binding but completely inhibited degradation of the hormone. Monensin, a carboxylic ionophore which impairs uncoupling hormone-receptor complexes, effectively restricted insulin degradation over short time periods (less than 30 min). Addition of monensin to insulin-stimulated cells did not impair D-glucose uptake. It has previously been reported that PAO inhibits hexose transport through the direct interaction with the glucose transporters and low concentrations of PAO (1 microM) transiently inhibit insulin-stimulated glucose uptake. This recovery phenomenon was again observed when PAO was added to insulin-stimulated, monensin-treated adipocytes. The data suggests that lysosomal degradation of insulin is not requisite for signal transduction.     

Characterization of a phosphate transport system in human fibroblast lysosomes.

The uptake of [32P]KH2PO4 by Percoll-purified human fibroblast lysosomes at pH 7.0 was investigated to determine if lysosomes contain a transport system recognizing phosphate. Lysosomal phosphate uptake was linear for the first 2 min, attained a steady state by 8-10 min at 37 degrees C, and was not Na+ or K+ dependent. Upon entering lysosomes, [32P]phosphate was rapidly metabolized to trichloroacetic acid-soluble and trichloroacetic acid-insoluble products. After 1-min incubations, 50% of the radioactivity recovered from lysosomes was in the form of inorganic phosphate; and after a 2.5-min incubation, 27% of the radioactivity was recovered as inorganic phosphate. When lysosomes are loaded with radioactivity by incubation with 0.03 mM [32P]KH2PO4 for 25 min and then washed at 4 degrees C, lysosomes fail to release the accumulated radioactivity during a subsequent incubation at 37 degrees C. Lysosomal phosphate uptake gave linear Arrhenius plots (Q10 = 1.8) and was inversely proportional to medium osmolarity. Phosphate uptake was maximal at pH 5-6, half-maximal at pH 7.1, with little transport activity at pH greater than 8, suggesting that the transport system recognizes the monobasic form of phosphate. Lysosomal phosphate uptake is saturable, displaying a Km of 5 microM at pH 7.0 and 37 degrees C. High specificity for phosphate is demonstrated since large concentrations of Na2SO4, NaHCO3, KCl, NaCl, 5'-AMP, or the anion transport inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, have no effect on lysosomal phosphate transport. In contrast, the phosphate analog, arsenate, strongly inhibits lysosomal phosphate uptake in a competitive manner with a Ki of 7 microM. Pyridoxal phosphate, CTP, adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), and glucose 6-phosphate were found to be noncompetitive inhibitors of lysosomal phosphate uptake displaying Ki values of 80-250 microM. When lysosomes are incubated with [gamma-32P]ATP, the lysosomal membrane ATPase hydrolyzes the ATP to form inorganic phosphate which then enters lysosomes by this lysosomal phosphate transport route.     

Nuclear translocation of the insulin receptor. A possible mediator of insulin's long term effects

The translocation of occupied surface insulin receptors to the nuclei of isolated hepatocytes was studied using the biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). When hepatocytes were photolabeled at 4 degrees C, extensively washed, and then further incubated at 37 degrees C for 1 h, photolabeled insulin receptors, which were initially localized to the cell surface, accumulated in the subsequently isolated nuclei. When the isolated nuclei were solubilized and subjected to polyacrylamide gel electrophoresis and radioautography, labeled proteins with Mr identical to the cell surface insulin receptor were detected. Light microscopic radioautography of nuclei isolated from cells incubated for 1 ha at 37 degrees C demonstrated that 28% of these nuclei were specifically labeled with one or more grains. Electron microscopic radioautography of intact cultured hepatocytes, incubated 60 min at 37 degrees C, revealed that 26% of the thin-sectioned nuclei contained at least a single grain and 8.3% of the total cell-associated associated grains were located over the nuclei. Only 1.6% of grains were localized to lysosomes. In contrast, if photolabeled hepatocytes were incubated at 4 degrees C for up to 2 h, negligible accumulation of nuclear radioactivity was observed by polyacrylamide gel electrophoresis on light or electron microscopic radioautography. Conclusions are as follows. Occupied cell surface insulin receptors can internalize and translocate to the nucleus of intact hepatocytes by a time- and temperature-dependent mechanism. Accumulation and possible degradation of insulin receptors in lysosomes involves only a small percentage of the receptors internalized. Nuclear translocation of occupied cell surface insulin receptors may be a mechanism which mediates insulin's long term effects.     

Binding and processing of (125)I-ACTH by isolated rat splenic lymphocytes

The effect of incubation temperature and ligand competition was tested for (125)I-ACTH binding to isolated rat lymphocytes. AlphaMSH but not Agouti-like peptide was an effective competitive inhibitor for cell surface binding at 4 degrees C. Cells incubated with (125)I-ACTH at 37 degrees C rapidly associated ligand for 10 min and then gradually lost the radioactivity with time. Cells incubated with (125)I-ACTH at 4 degrees C accumulated ligand to only about half the maximal amount when compared to cells incubated at 37 degrees C for 10 min. Temperatures below 20 degrees C and toxins that block lysosomal degradation blocked the loss of cell-associated radioactivity. These results suggest the lymphocyte ACTH receptor is the Melanocortin 5 receptor and the receptor is internalized by endocytosis to deliver ligand to the lysosome.     

Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line.

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Amino acid transport by aggregates of cultured chicken heart cells. Effect of insulin.

Single heart cells dissociated from 14-day-old chicken embryos were reagregated into spheroidal clusters on a gyratory shaker and centrifuged to form cohesive discs of approximately 400 aggregates. These cultured cells accumulated 2-amino[1-14C]isobutyric acid against a gradient. When incubated for 3 hours in a protein-free, buffered, balanced salt solution, concentrative transport decreased to a stable basal level. Incubation in the presence of sodium bovine insulin prevented this fall in transport activity and resulted in increased 2-aminoisobutyric acid uptake to concentrations 40 time sthat in the medium during a subsequent 3-hour transport assay. Intracellular accumulation of 2-aminoisobutyric acid was linear during the initial 15 min of transport in the presence and absence of added insulin. Basal transport of 2-aminoisobutyric acid was temperature-dependent, requied extracellular sodium greater than 125 meg/liter, and demonstrated saturation with an apparent Vmax of 3.4 mmol/liter/10 min and an apparent Km of 2.6 mM. Basal transport activity was not reduced by cycloheximide or puromycin even after 3 hours of exposure...     

Stimulation of 3-O-methylglucose transport by anaerobiosis in rat thymocytes.

Transport of 3-O-methylglucose by rat thymocytes occurs by facilitated diffusion and follows a biphasic time course. The half-times of the two phases of uptake are 0.8 min and 20 to 30 min; the rapid phase contributes 10 to 20% of the total 3-O-methylglucose taken up at equilibrium. Cells incubated under anaerobic conditions for 1 hour undergo a 3- to 4-fold increase in the initial rate of 3-O-methylglucose uptake. The relative contribution of the rapid phase of uptake increases nearly 4-fold in anaerobically incubated cells, although the half-time of the rapid phase remains the same. Anaerobiosis also reduces the half-time of the slow phase of uptake by a factor of three. In the absence of exogenous glucose, anaerobiosis reduces cellular ATP by 97% after 1 hour at 37 degrees. However, full stimulation of transport activity does not occur in cells with such low levels of ATP. When anaerobically incubated cells are re-exposed to oxygen, ATP synthesis proceeds and transport activity increases by 100% within 5 to 10 min. Adding 1 mM 2,4-dinitrophenol at the time the anaerobic cells are reexposed to oxygen completely blocks the subsequent ATP synthesis and the associated increase in transport activity. Cells incubated aerobically in the presence of 1 mM 2,4-dinitrophenol show a 90% reduction in ATP levels and a 2-fold increase in the rate of 3-O-methylglucose uptake. An additional 70% increase in transport activity is observed when the cells are washed free of uncoupler and incubated an additional 10 min. The results suggest that transport activity is stimulated when cellular ATP levels decline but that the stimulation process requires some minimal level of ATP for full expression.     

Effect of anti-insulin receptor antibodies on insulin-sensitive phosphodiesterase in rat fat cells

Effect of sera with anti-insulin receptor antibodies (AIRS) on insulin-sensitive phosphodiesterase in rat fat cells was examined. AIRS activated the enzyme when incubated with intact fat cells. AIRS (1:400 dilution) were less potent for activation of the phosphodiesterase than insulin (3 nM), but were more potent for inhibition of 125I-insulin binding to fat cells than insulin. When insulin receptor of fat cells was destroyed with trypsin-treatment, AIRS as well as insulin completely lost the ability to activate the phosphodiesterase. These findings suggest that AIRS bind to or very near the insulin receptor and exhibit insulin-like biological effect of the phosphodiesterase activation.     

Effects of okadaic acid on insulin-sensitive cAMP phosphodiesterase in rat adipocytes. Evidence that insulin may stimulate the enzyme by phosphorylation.

Okadaic acid, a potent inhibitor of Type 1 and Type 2A protein phosphatases, was used to investigate the mechanism of insulin action on membrane-bound low Km cAMP phosphodiesterase in rat adipocytes. Upon incubation of cells with 1 microM okadaic acid for 20 min, phosphodiesterase was stimulated 3.7- to 3.9-fold. This stimulation was larger than that elicited by insulin (2.5- to 3.0-fold). Although okadaic acid enhanced the effect of insulin, the maximum effects of the two agents were not additive. When cells were pretreated with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), the level of phosphodiesterase stimulation by okadaic acid was rendered smaller, similar to that attained by insulin. In cells that had been treated with 2 mM KCN, okadaic acid (like insulin) failed to stimulate phosphodiesterase, suggesting that ATP was essential. Also, as reported previously, the effect of insulin on phosphodiesterase was reversed upon exposure of hormone-treated cells to KCN. This deactivation of previously-stimulated phosphodiesterase was blocked by okadaic acid, but not by insulin. The above KCN experiments were carried out with cells in which A-kinase activity was minimized by pretreatment with H-7. Okadaic acid mildly stimulated basal glucose transport and, at the same time, strongly inhibited the action of insulin thereon. It is suggested that insulin may stimulate phosphodiesterase by promoting its phosphorylation and that the hormonal effect may be reversed by a protein phosphatase which is sensitive to okadaic acid. The hypothetical protein kinase thought to be involved in the insulin-dependent stimulation of phosphodiesterase appears to be more H-7-resistant than A-kinase.     

Cell surface receptor for hemopexin in human leukemia HL60 cells. Specific binding, affinity labeling, and fate of heme

The binding of 125I-labeled human hemopexin to human leukemia HL60 cell at 4 degrees C was saturable with time and with increasing concentrations of 125I-hemopexin. Scatchard analysis of the binding data revealed the presence of approximately 42,000 binding sites/cell with an apparent dissociation constant (Kd) of 1.0 X 10(-9) M. When cells were incubated with radioactive hemopexin at 37 degrees C, 125I-hemopexin was rapidly bound and then was dissociated after the release of heme. Treatment of surface-bound 125I-hemopexin with divalent lysine-directed cross-linking disuccinimidyl suberate revealed a membrane polypeptide of about 80,000 Da, to which hemopexin is cross-linked. To examine the fate of the internalized heme, lysates from the cells previously incubated with [59Fe]heme-hemopexin complex were analyzed by CM-cellulose and Sephacryl S-200 column chromatography. A considerable amount of the radioactivity was present in the fraction which co-eluted with the myeloperoxidase activity. When myeloperoxidase was isolated from the cells incubated with [59Fe]heme-hemopexin complex by immunoprecipitation with anti-myeloperoxidase antibody, radiolabeled iron associated with myeloperoxidase increased with time, and more than 30% of the radioactivity in the cells was present in the myeloperoxidase. These results indicate that the binding of hemopexin to the surface receptors triggers a release of heme and that this heme is incorporated into the intracellular myeloperoxidase.     

Receptor-linked degradation of 125I-insulin is mediated by internalization in isolated rat hepatocytes

When hepatocytes were freshly isolated from rat liver and incubated for various periods of time at 37 degrees C, the media from the incubation, when completely separated from the cells, actively degraded 125I-insulin. THis soluble protease activity was strongly inhibited by bacitracin but was unaffected by the lysosomatropic agent ammonium chloride (NH4Cl). When hepatocytes were incubated with 125I-insulin at 37 degrees C in the presence or absence of 8 mM NH4Cl the ligand initially bound to the plasma membrane and was subsequently internalized as a function of time. When hepatocytes were incubated at 37 degrees C for 30 minutes with 125I-insulin in the presence of bacitracin and NH4Cl or bacitracin alone and the cells were washed, diluted, and the cell-bound radioactivity allowed to dissociate, the percent intact 125I-insulin in the cell pellet and in the incubation media was greater in the presence of NH4Cl at each time point of incubation. Under these same conditions a higher proportion of the cell-associated radioactivity was internalized and a higher proportion was associated with lysosomes. The data suggest that receptor-mediated internalization is required for insulin degradation by the cell, and that this process, at least in part, involves lysosomal enzymes. Furthermore, the data demonstrate that internalization is not blocked by the presence of bacitracin or NH4Cl in the incubation media, but that degradation is inhibited.     

Catecholamine-induced insulin resistance of glucose transport in isolated rat adipocytes.

The effects of pre-incubation with isoprenaline and noradrenaline on insulin binding and insulin stimulation of D-glucose transport in isolated rat adipocytes are reported. (1) Pre-incubation of the cells with isoprenaline (0.1-10 microM) in Krebs-Ringer-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid] buffer (30 min, 37 degrees C) at D-glucose concentrations of 16 mM, in which normal ATP levels were maintained, caused a rightward-shift in sensitivity of D-glucose transport to insulin stimulation by 50% and a decrease in maximal responsiveness by 30% (2) [A14-125I]insulin binding was reduced significantly by 35% at insulin concentrations less than 100 mu-units/ml and Scatchard analysis showed that this consisted mainly of a decrease in high-affinity binding. (3) Pre-incubation with catecholamines under the same conditions but at low glucose concentrations (0-5 mM) caused a fall in intracellular ATP levels of 65 and 45% respectively. (4) The fall in ATP additionally lowered insulin binding by 50% at all insulin concentrations and a parallel shift of the binding curves in the Scatchard plot showed that this was due to a decrease in the number of receptors. (5) At low and high ATP concentrations the insulin stimulation of D-glucose transport was inhibited to a similar extent. (6) Pre-incubation with catecholamines thus inhibited insulin stimulation of D-glucose transport in rat adipocytes mainly by a decrease in high-affinity binding of insulin, which was not mediated by low ATP levels. This mechanism may play a role in the pathogenesis of catecholamine-induced insulin resistance in vivo.     

Inhibition of metabolic effects of growth hormone by various inhibitors of cyclic nucleotide phosphodiesterase

When isolated diaphragms of hypophysectomized rats were incubated with bovine growth hormone in the presence of the cyclic nucleotide inhibitors theophylline, quinine and papaverine, the stimulatory effects of the hormone on leucine incorporation into protein, α-aminoisobutyric acid and 3-O-methylglucose transport were suppressed or abolished entirely. The degree of suppression of the hormone effects appeared to correlate with the extent of glycogenolysis caused by the drugs. Thoephylline also rapidly reversed the stimulation of protein synthesis and amino acid and sugar transport produced by growth hormone. When protein synthesis and transport were stimulated by preincubation of the diaphragm with growth hormone, the subsequent addition of theophylline to the medium inhibited the hormonal effects on protein synthesis and sugar transport within 15 min and the effect on amino acid transport within 60 min. These results may mean that the rapid in vitro effects of growth hormone on protein synthesis and membrane transport in rat diaphragm muscle are mediated by a reduction in the cellular level of cyclic AMP or some other nucleotide.Attempts to block the action of growth hormone on 3-O-methylglucose transport by preincubation of the diaphragm with high concentrations (10 mM) of cyclic GMP, cyclic UMP, cyclic TMP and cyclic CMP were unsuccessful. Also an effort was made to mimic the action of growth hormone on sugar transport by incubating the diaphragm with high concentrations of imidazole and histamine, agents known to activate cyclic nucleotide phosphodiesterase. Slight stimulatory effects were obtained, but they could not be correlated with any certainty to the actions of imidazole and histamine on phosphodiesterase.Like growth hormone, insulin also stimulates protein synthesis and amino acid and sugar transport in the isolated rat diaphragm. However, the actions of insulin on these processes were not abolished by theophylline, suggesting some basic difference in the mode of action of these two hormones on protein synthesis and membrane transport in muscle.     

Inhibitory effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport and on 125I-labelled insulin binding by rat soleus muscle

These experiments examined the effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport in soleus muscle in terms of the Thiol-Redox model for insulin-stimulated adipocyte sugar transport (Czech, M.P. (1976) J. Cell. Physiol. 89, 661-668). Brief exposure (1 min) to N-ethylmaleimide (0.3-10 mM) inhibited the stimulatory effect of insulin (0.1 U/ml) on D-[U-14C]xylose uptake by rat soleus muscle. N-Ethylmaleimide also inhibited the stimulatory effects of H2O2 (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin action was paralleled by the inhibition of 125I-labelled insulin binding by the muscle. N-ethylmaleimide lowered muscle ATP; however, its effects on sugar transport and 125I-labelled insulin binding could be dissociated from its effect on ATP. Exposing muscles to insulin prior to N-ethylmaleimide did not abolish the inhibitory effect of sulphydryl blockade on insulin-stimulated sugar transport, but did reduce the effect of the inhibitor by 20-30%. Conversely, when muscles were first allowed to bind 125I-labelled insulin and then exposed to the inhibitor, there was no effect of N-ethylmaleimide on pre-bound insulin. Exposure to diamide or vitamin K-5 before N-ethylmaleimide (1 mM) attenuated the inhibitory effect of sulphydryl blockade but no protective effect was observed with H2O2. None of the oxidants protected against the inhibitory effect of 3 mM N-ethylmaleimide. It is concluded that there are two N-ethylmaleimide-sensitive sites involved in the activation of muscle sugar transport at the post-receptor level. One of these would appear to be similar to the Thiol-Redox site described in the adipocyte; the other site appears to be an essential sulphydryl group whose function does not involve oxidation to a disulphide.