The GIP receptor on pancreatic beta cell tumor: molecular identification by covalent cross-linking

125I-GIP binds reversibly to a high affinity binding site in crude plasma membranes prepared from a hamster pancreatic beta cell tumor. The treatment of labeled membranes with the cross-linker dithiobis (succinimidylpropionate) prevents, to a greater extent, the rapid dissociation of 125I-GIP-membrane complexes which is observed when 10(-6) M native GIP is added. Polyacrylamide gel electrophoresis of membrane proteins reveals a major 125I-GIP-protein complex of Mr 64,000. This labeling decreases when increasing concentrations (10(-9) -10(-6)M) of native GIP are added but is not altered by other peptide hormones (tested at 10(-6)M) including glucagon, VIP and insulin. The Mr 64,000 complex is not observed in tissues which have no specific binding sites for GIP such as intestinal epithelium. Assuming one molecule of 125I-GIP is bound per molecule of protein, one protein with Mr 59,000 is identified as the specific GIP binding site.     

Role of ATP in specific binding of 125I-insulin to cytoplasmic receptors of the liver and muscle membranes of controls and diabetic rats

A study was made of the action of various concentrations of ATP on insulin ability to bind to the receptors of the liver and muscle membranes in control and streptozocin-induced diabetes animals. Specific binding of 125I-insulin to the receptors of the liver and muscle membranes was shown to rise in animals with streptozocin-induced diabetes as compared to control. This effect was most pronounced in the muscle membranes. Preincubation of the membranes with ATP did not affect insulin binding to the liver and muscle receptors of control animals. However, hormone binding to the liver receptors of diabetic rats was drastically suppressed by ATP (10(-3) M). Less ATP concentrations (10(12) M) produced an additional inhibitory action which was not marked. ATP led to decreased insulin binding to the muscle receptors of diabetic rats only at extremely low concentrations (10(-12) M). The data obtained may be of importance for regulation of membrane phosphorylation in the states characteristic of insulin resistance.     

Effects of insulin on the lipid structure of liver plasma membrane measured with fluorescence and ESR spectroscopic methods

Insulin increased the lipid order of rat and mouse liver plasma membrane domains sampled by the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in a concentration-dependent saturable manner. The ordering is half maximal at $\text{5.1 · 10}{}^{\mathrm{?11}}\text{M}$ and fully saturated at $\text{1.7 · 10}{}^{\mathrm{?10}}\text{M}$ insulin. Membranes prepared from obese hyperglycemic (ob / ob) mice demonstrated a right-shift in the dose-dependent ordering induced by insulin, such that ordering was half maximal at $\text{1.2 · 10}{}^{\mathrm{?10}}\text{M}$ and fully saturated at $\text{2.0 · 10}{}^{\mathrm{?10}}\text{M}$. Insulin also increased the order of rat liver plasma membranes labeled with the cis- and trans-parinaric acid methyl esters. The ordering caused by insulin as detected with cis methyl parinarate was complete within approx. 15 min. after hormone addition at 37°C, and the ordering was approximately double that observed with the trans isomer. Additional ESR experiments demonstrated that the addition of insulin increased the outer hyperfine splittings of spectra recorded from membranes labeled with the steroid-like spin labels, nitroxide cholestane and nitroxide androstane, but not the fatty acid spin probe, 5-nitroxide stearate. Studies utilizing model membrane systems strongly suggest that the 5-nitroxide stearate samples a cholesterol-poor domain of the membrane, while the steroid-like probes preferentially sample cholesterol-rich regions of the membrane. Finally, insulin-induced membrane ordering was dose-dependently inhibited by cytochalasin B in the range 1–50 μM. From these results, we conclude that (1) the ordering effect of insulin addition to isolated liver plasma membrane fractions occurs within the physiological range of hormone concentration, and the dose-response is right-shifted in membranes from ‘insulin resistant’ animals; (2) the relative responses of the fluorescent and spin probes suggest that the effects of insulin are confined to specific domains within the membrane matrix; and (3) the direct effects of insulin on the membranes may involve protein components having cytochalasin B binding sites.     

Characterization of insulin binding to bovine liver and mammary microsomes

Bovine liver and mammary gland (MG) appear metabolically independent of insulin, yet the specificity and kinetics of 125I-insulin (125I-INS) binding to bovine liver and MG microsomes (MIC) indicate the presence of insulin receptors in MIC from both tissues. The insulin receptors from bovine liver (Kd = 7.6 X 10(-10) M) and MG (Kd = 9.6 X 10(-11) M) were similar to each other and to other insulin receptors in their binding affinities and pH optima. Perturbation of rat liver and bovine MG MIC by phospholipase or NaCl treatment increased 125I-INS binding to the membranes, suggesting exposure of cryptic insulin receptors. Different responses in 125I-INS binding to membrane perturbation suggest differences between rat and bovine membranes.     

Effect of bovine growth hormone on rat liver plasma membranes as studied by circular dichroism and fluorescence using the extrinsic probe 7,12-dimethylbenzanthracene

Conformational changes produced by in vitro bovine growth hormone addition to plasma membranes of hypophysectomized rat liver proteins and lipids have been studied by circular dichroism as well as intrinsic and extrinsic fluorescence. 7,12-Dimethylbenzanthracene has been used as a fluorescent probe of changes in membrane structure. The exposure of membranes to bovine growth hormone produced a change in membrane negative ellipticity. Dimethylbenzanthracene at concentrations similar to those employed in fluorescence studies had no effect on the membrane circular dichroism spectrum. Its presence did, however, prevent a response to growth hormone. There was a decrease in peak fluorescence intensity and a peak shift when bovine growth hormone (0.5 · 10^?12 M) was added to liver membranes. The addition of dimethylbenzanthracene (1.6 · 10^?6 M) to membranes resulted in a decrease in the intensity of the protein fluorescence peak at 335 nm and the appearance of two peaks at 430 and 407 nm, assignable to the probe. The addition of bovine growth hormone (0.5 · 10^?12 M) produced a decrease in fluorescence at 335 nm and also in the peaks at 407 and 430 nm. These data are consistent with the conclusion that bovine growth hormone produces a conformational change in rat liver plasma membrane proteins and lipids.     

Comparison of Insulin Binding Proteins In Plasma and Nuclear Membranes of Obese and Lean Mouse Liver

Abstract

Plasma membranes obtained from obese (ob/ob) and lean (+/+ or +/ob) mouse livers were chemically crosslinked to [¹²⁵I] -insulin and examined by electrophoresis and autoradiography. The pattern of crosslinked hormone was qualitatively similar in obese and lean plasma membranes. A major insulin binding protein of approximately M 120,000 was observed. Two additional bands were apparent, one which remained near the top of the gel and one about M 90,000. A minor band at approximately M 50,000 was also detected. For each of the insulin binding proteins a reduction in the amount of [¹²⁵I]-insulin bound was observed with obese plasma membranes as compared with lean. For all proteins the insulin binding was specific as determined by competition with unlabeled hormone. In addition to plasma membrane receptors, insulin has also been reported to bind to nuclear membranes. The autoradiographic patterns of gels of [¹²⁵]-insulin bound and crosslinked to nuclear membranes from obese and lean mouse livers indicated the presence of proteins of the same M as those described for plasma membranes. Nuclear membrane proteins bound less insulin than plasma membranes and, again, the obese was decreased relative to the lean. Contamination of the nuclear membrane fraction by plasma membranes was ruled out. Scatchard analyses of [¹²⁵]-insul in bound to plasma and nuclear membranes indicated that the decrease in hormone binding in the obese mouse is a result of a reduction in the absolute number of receptors. The findings presented in this study provide additional support for this conclusion by demonstrating that membranes from obese mice are comprised of the same set of apparently unaltered insulin binding proteins. Further, the presence of similar insulin binding proteins in both nuclear and plasma membranes suggests a physiological relationship between these structures with respect to hormone binding and/or in the mechanism of action of insulin.     

Comparative studies on the major features of insulin receptors in mammalian and non-mammalian liver membranes

1. The camel has insulin receptors that by multiple function criteria are very similar to those of the other mammals (rabbit and rat) and non-mammals (chicken and pigeon), with sharp pH dependence to insulin binding at pH 7.2-7.6. 2. Equilibrium binding was faster at higher temperatures (24-37 degrees C) than at lower (4 degrees C). 3. Binding data yielded curvilinear Scatchard plots with half maximal displacement of 125I-insulin at 9 x 10(-9) M, 2.5 x 10(-9) M, 6.3 x 10(-10) M for camel, rabbit, pigeon and chicken respectively, suggesting differences in mammalian and non-mammalian liver membranes. 4. Autoradiogram patterns showed the presence of an identical subunit structure with Mr 74,000 for all membranes studied. Pigeon membrane showed a band with Mr 110,000, the absence of which in other membranes could be due to the degradation factor or the concentration of disuccinimidyl suberate (DSS).     

The mechanisms of up-regulation of the hepatic insulin receptor in hypoinsulinemic diabetes mellitus

The mechanism underlying the increased insulin binding found in hepatic plasma membranes from streptozotocin-diabetic rats was evaluated by measuring insulin binding to intact and Triton X-100-soluble extracts of plasma membranes prepared from the livers of control rats and rats administered streptozotocin (85 mg/kg). In addition, to assess whether the cellular content of hepatic insulin receptors is also increased in diabetic animals, we measured insulin-binding activity in intact and soluble extracts of total hepatic cellular membrane preparations (100,000 X g cellular pellets). The data indicate that while insulin binding is increased (52 +/- 3%) in intact hepatic plasma membranes from diabetic rats compared to control rats, there is no comparable increase in insulin binding in intact total cellular membranes or in Triton X-100-soluble extracts of plasma membranes or total cellular membranes. We therefore conclude that the enhanced insulin binding found in the livers of diabetic rats is the result of a local redistribution of plasma membrane insulin receptors from cryptic to exposed sites. Finally, the data suggest the presence of a negative modulator of insulin-binding affinity in intact plasma and total cellular membranes.     

Involvement of calcium in the inhibition by insulin of the glucagon-stimulated adenylate-cyclase activity.

1. The inhibitory effect of adenosine on the glucagon-stimulated adenylate cyclase activity of liver plasma membranes, prepared from PVG/c rats, was potentiated by insulin. In the presence of EGTA, such potentiating effect of insulin was lost. 2. Calcium (10 microM) potentiated the inhibitory effects of both adenosine and insulin on the glucagon-stimulated cyclase activity. The synergestic effect of calcium + insulin required the presence of adenosine as judged from the use of adenosine deaminase. 3. Insulin had no significant inhibitory effect on the glucagon-stimulated cyclase activity of liver plasma membranes, prepared from young Wistar rats, unless both adenosine (50 microM) and calcium (10 microM) were added externally. 4. Results demonstrate an interaction of calcium and insulin at membrane level that, in the presence of adenosine, results in the inhibition of the glucagon-stimulated adenylate cyclase activity.     

Study of insulin-receptor interactions in plasma membrane of rat liver using antibodies against insulin

Preincubation of rat liver cell plasma membranes with antiinsulin serum (AIS) results in a 3-fold increase of specific binding of [125I]insulin added to AIS-free membranes. The degree of such increase depends on preincubation time and AIS concentration; at a rise of both parameters the dependence curve shown a plateau. The Scatchard plots suggest that preincubation with AIS increases the high and low affinity of binding sites; their number remains thereby unchanged. Using several types of immune and non-immune sera for preincubation the specificity of this effect was established. It was assumed that the antibodies initiate the immunological reaction of the membrane. Study of AIS effect on dissociation of membrane-bound labelled insulins differing in their affinities for the given AIS revealed that the antibodies can interact with the newly formed hormone-receptor complex but cannot affect the receptor within insulin. It is concluded that the intact hepatocyte membrane contains a receptor-bound insulin which is a target of AIS action. The antigen-antibody interaction changes the functional state of occupied and free receptors which bind [125I]insulin. The latter becomes repeatedly involved in this process as a result of destabilization of site-site interactions provoked by antibodies against the hormones.     

An insulin-stimulated cation channel in skeletal muscle. Inhibition by calcium causes oscillation.

A cation channel has been identified in the plasma membrane of skeletal muscle that oscillates open and closed in a regular manner. In an experimental system of patch-clamped reconstituted plasma membrane in phospholipid bilayers, the oscillations are calcium-dependent and constitute regular closing events due to inhibition of the channel by calcium with a Ki of 2.2 +/- 1 x 10(-6) M, followed by reopening. There are 3.7 +/- 1 calcium binding sites/channel. With sodium as the current vehicle, conductance is increased by voltage, insulin (Km = 5 +/- 0.6 x 10(-9) M), and hydrolyzable guanine nucleotides. Cyclic GMP alone with increase the conductance with a Km of 3.7 +/- 0.6 x 10(-7) M. In the absence of calcium, the unitary conductance with insulin + GTP or cGMP at 150 mM NaCl is 153 picosiemens. Sodium current is insensitive to 10(-5) M tetrodotoxin but inhibited by mu-conotoxin (Ki = 5 x 10(-8) M). These findings in the reconstituted system were verified in patch-clamped whole muscle cells where an insulin and cGMP-dependent sodium current inhibited by mu-conotoxin could be demonstrated. In the whole cell experiments, slow calcium-dependent oscillations of the sodium current were also detected.     

Calcium modulates fatty acid dynamics in rat liver plasma membranes

Modulation of free fatty acid binding in isolated rat liver plasma membranes was evaluated using the fluorescent fatty acids trans-parinaric and cis-parinaric acid as analogues for saturated and unsaturated fatty acids, respectively. Binding of trans-parinarate but not cis-parinarate was inhibited by physiological levels of Ca2+. The effect was reversed by addition of excess EGTA. Calcium decreased the aqueous to lipid partition coefficient, Kp, of trans-parinaric acid for liver plasma membranes while increasing the Kp for cis-parinaric acid. In addition, Ca2+ also altered the fluorescence lifetime, the quantum yield, and the relative partitioning of trans-parinaric and cis-parinaric acid into fluid and solid phases. Calcium and EGTA did not affect the binding of 1,6-diphenyl-1,3,5-hexatriene. The effect of Ca2+ on the liver plasma membrane structure was to increase the rigidity of the membrane, primarily the solid domain. The fluorescence polarization of trans-parinarate, cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene at 24 degrees C in liver plasma membranes in the absence of Ca2+ was 0.295 +/- 0.008, 0.253 +/- 0.007, and 0.284 +/- 0.005, respectively. Calcium (2.4 mM) increased the polarization of these probe molecules in liver plasma membranes by 8-10%. EGTA (3.4 mM) reversed or abolished the increase in polarization. Thus, the fluorescent fatty acids trans-parinarate and cis-parinarate may be used to monitor fatty acid binding by isolated membranes, to evaluate factors such as Ca2+ which modulate fatty acid binding, and to investigate the microenvironment in which the fatty acids residue. The data suggest that Ca2+ may be an important regulator of fatty acid uptake by the liver plasma membrane, and thereby interact with intermediary metabolism of lipids at a step not involving lipolytic or synthetic enzymes.     

Cellular binding sites for insulin in rat liver.

A study of the sites of insulin binding in subcellular fractions of rat liver is reported. A method for the isolation of liver plasma membranes, which permits one to follow quantitatively the distribution of all the parameters of interest, was modified and applied to the study of the cellular topography of insulin binding. The insulin binding capacity did not follow closely the enzyme marker (5'-nucleotidase) for plasma membranes when differential centrifugation schemes were used, and the divergence from this marker was more prominent when separations were performed on discontinous sucrose gradients. A significant amount of insulin binding capacity was always present in fractions with higher density than those containing the majority of 5'-nycleotidase. Results of studies on linear sucrose gradients have disclosed in some of the purified membrane fractions small but consistent differences in density of the insulin binding, and plasma membrane particles. It is suggested that there may be several types of intracellular membranes to which insulin can bind besides the plasma membranes.     

Purification and properties of a peptic heme peptide from cytochrome c1.

Highly purified mouse liver plasma membranes have been used to define the properties of an NADH dehydrogenase activity associated with plasma membrane. The NADH indophenol reductase activity is two-fold stimulated at 5 × 10^?8 M glucagon and the stimulation is inhibited by atebrin. Corresponding activity in endoplasmic reticulum is not stimulated by glucagon. The NADH indophenol reductase is 90% inhibited by insulin at 7 × 10^?11M and shows a return to the original activity at higher insulin concentrations. NADH dehydrogenase activity in endoplasmic reticulum is inhibited up to 50% by insulin at a similar concentration. Triiodothyronine at 10^?7M also inhibits the plasma membrane dehydrogenase whereas thyroxine has little effect. The response of this dehydrogenase to hormones suggests a role in regulation of cellular function.     

Insulin receptors prepared with iodoacetamide show enhanced autophosphorylation and receptor kinase activity.

In this study, we found that adding iodoacetamide to the homogenization buffer used in the preparation of mouse or rat liver plasma membranes resulted in an increase of insulin receptor autophosphorylation by 4-5-fold and receptor kinase activity by about 2-fold. Similar effects were obtained with iodoacetate and p-chloromercuriphenyl sulfonate. The effect of iodoacetamide was minimal when it was added to membranes prepared without the thiol reagent. The enhancing effect of iodoacetamide on insulin receptor autophosphorylation was the result of a more than 2-fold decrease in the Km and a more than 3-fold increase in Vmax for ATP. The presence of iodoacetamide in the preparation of plasma membranes also greatly increased the solubilization of the insulin receptor from the plasma membrane by Triton X-100. We propose that iodoacetamide acts to alkylate some unknown thiols released during tissue homogenization and that in its absence these thiols formed mixed disulfides with the insulin receptor, thus adversely affecting the process of receptor activation by insulin.     

Identification of the d-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[³H]Cytochalasin B binding and its competitive inhibition by d-glucose have been used to identify the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [³H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for d-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the d-glucose-inhibitable site. If 280 nM (40 000 μunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of d-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Effect of isoflavone genistein on insulin receptors in perfused liver of ovariectomized rats

The experiments were carried out on ovariectomized Wistar rats. Their livers were perfused with basic perfusion medium (BPM) or BPM supplemented with isoflavone genistein, insulin or combination of the two factors. The obtained results support the hypothesis that genistein influences the kinetics of insulin binding to cell membranes changing the number of insulin receptors and dissociation constant (Kd). BPM supplementation with genistein decreased number of high affinity insulin receptors (HAIR) both in livers treated and untreated with insulin. The amount of HAIR diminished significantly from 610 +/- 77 x 10(-15) (no genistein) to 238 +/- 72 x 10(-15) mol/mg of membrane protein (supplement of genistein). Similarly, genistein reduced slightly the amount of HAIR even when added together with insulin (372 +/- 59 x 10(-15) mol/mg) in comparison to rats perfused with medium containing insulin but not the isoflavone (421 +/- 46 x 10(-15) mol/mg). Simultaneously, genistein decreased significantly Kd for HAIR (perfusion with BPM--1.44 +/- 0.18 x 10(-9) mol/l; perfusion with BMP + genistein--0.83 +/- 0.20 x 10(-9) mol/l). Such effects of genistein during liver perfision did not take place when the liver membranes were in vitro incubated with this xenobiotic.     

Cellular binding sites for insulin in rat liver

A study of the sites of insulin binding in subcellular fractions of rat liver is reported. A method for the isolation of liver plasma membranes, which permits one to follow quantitatively the distribution of all the parameters of interest, was modified and applied to the study of the cellular topography of insulin binding. The insulin-binding capacity did not follow closely the enzyme marker (5′-nucleotidase) for plasma membranes when differential centrifugation schemes were used, and the divergence from this marker was more prominent when separations were performed on discontinuous sucrose gradients. A significant amount of insulin binding capacity was always present in fractions with higher density than those containing the majority of 5′-nucleotidase. Results of studies on linear sucrose gradients have disclosed in some of the purified membrane fractions small but consistent differences in density of the insulin binding, and plasma membrane particles. It is suggested that there may be several types of intracellular membranes to which insulin can bind besides the plasma membranes.     

Insulin: interaction with membrane receprots and relationship to cyclic purine nucleotides and cell growth.

Insulin action is discussed with emphasis on events that occur at the plasma membrane. A summary is presented of previous studies which indicate that the insulin receptor of fat and liver cells is a large glycoprotein, partially buried in the outer surface of the plasma membrane, with a high (K-D approximately 10-10 M) and specific affinity for insulin. The participation of membrane phospholipids in the binding of insulin and the role of sialic acid residues in the transmission of the insulin binding signal are discussed. The relation of insulin action to its effects on cyclic nucleotide levels is explored. On the one hand, insulin action (glucose transport) is inhibited by compounds (cholera toxin, ACTH, glucagon and L-norepinephrine) that stimulate adenylate cyclase; conversely, insulin both inhibits the lipolytic action of these compounds, and raises cellular levels of cyclic GMP. An hypothesis is presented whereby a single cyclase species may be responsible for the formation of either cyclic AMP or cyclic GMP, depending on the nature of the hormone stimulus. The role of membrane phosphorylation in the action of insulin is discussed in the context of experiments demonstrating a specific inhibition by ATP of insulin-mediated glucose transport, in association with the phosphorylation of two specific membrane proteins. The ability of insulin to modulate cyclic nucleotide levels in cultured cells and to act as a growth factor is discussed. Insulin stimulates DNA synthesis and the uptake of alpha-aminoisobutyric acid in human fibroblasts, which effects are also mediated by epidermal growth factor. Insulin acts at concentrations much higher than those obtained in vivo, whereas epidermal growth factor acts at concentrations thought to be physiological. The insulin binding sites (K-D is approximately equal to 10-9 M) related to growth, and observed both in human fibroblasts and in lectin-stimulated and leukemic human lymphocytes would not be appreciably occupied at physiological insulin concentrations. The implications of such 'low affinity' binding sites for insulin are discussed in relation to the action of other growth factors.     

Cell surface proteolysis and down-regulation of the hepatic insulin receptor. Evidence for selective sorting of intact and degraded receptors after internalization

Insulin binding to rat liver plasma membranes promotes proteolysis of the Mr 135,000 alpha subunit of the insulin receptor to a fragment of Mr 120,000 (Lipson, K. E., Yamada, K., Kolhatkar, A. A., and Donner, D. B. (1986) J. Biol. Chem. 261, 10833-10838). The enzyme that catalyzes this degradation copurifies with plasma membranes and cannot be identified in any other cellular organelle or in cytosol. The proteinase has optimal activity above pH 7 and is an integral protein based upon its resistance to extraction with 2 M NaCl. After affinity labeling, degraded insulin receptors were identified in plasma membranes isolated from a liver perfused with 1 nM 125I-insulin for 10 min at 37 degrees C, indicating that proteolysis occurs in the hepatocyte cell membrane under physiological conditions. Microsomes do not contain the receptor degrading activity or a detectable amount of degraded receptors under basal conditions. After perfusion of a liver with 125I-insulin, Mr 135,000 and Mr 120,000 complexes were detected in microsomes, suggesting that both intact and degraded receptors can be internalized. The initial absence of degraded receptors in plasma membranes suggests that, following internalization, such sites do not recycle. Thus, hormone-induced proteolysis of the insulin receptor begins at the surface of the rat hepatocyte and can lead to loss of receptors from the plasma membrane.