Irreversible Antagonism of Beta-Adrenoceptors with Para-Amino-Benzyl-Carazolol Provides Further Evidence for an Atypical Rat Adipocyte Beta-Adrenoceptor

Abstract

Rat adipocytes possess typical beta₁ adrenoceptors that can be identified by ¹²⁵I-cyanopindolol binding but the receptor mediating isoprenaline adenylate cyclase activation possesses properties quite unlike beta₁ or beta₂ receptors. Separation of these sites has been attempted using the photoaffinity antagonist para-amino-benzyl-carazolol. Preincubation of rat reticulocyte and adipocyte membranes with this agent followed by washing induced a concentration-dependent loss of specific ¹²⁵I-cyanopindolol sites in both tissues, though the maximal loss was apparently greater in the reticulocyte. However, the loss of sites in both tissues induced a different effect on isoprenaline-stimulated adenylate cyclase. In the reticulocyte, the loss of specific sites was accompanied by an equivalent fall in the maximal stimulation of adenylate cyclase. In the adipocyte there were no significant effects of receptor site loss on the isoprenaline dose-response curve. It is suggested that this data supports the concept that an atypical beta-adrenoceptor, with relatively low affinity for many antagonists, mediates catecholamine-stimulated adenylate cyclase (and lipolysis) in the adipocyte.     

Antibody Bingling Boyding to the Juxtamembr are Ahch of the Insulin Receptor Alters Reotr Affinity

Abstract

The effect of three antibodies that interact with distinct regions of the insulin receptor (the a subunit (83-7), the juxtamembrane region near tyrosine 960 (960) or the carboxy terminal region of the I3 subunit (CT-1)) on insulin binding was examined. Detergent-solubilized insulin receptors from IM-9 cells immobilized on Sepharose beads by 960 antisera bound 2-3 times more IWinsulin tracer (25-60 pM) than receptors immobilized with either 83-7 or CT-1. &-incubation of solubilized receptors with either 83-7 or 960 resulted in equivalent depletion (90%) of insulin binding activity from solubilized IM-9 cell extracts, suggesting that both antibodies were in excess and capable of binding a similar population of receptors. Antibody 960, but not CT-1 or 83-7, also increased insulin binding 2 fold to solubilized receptors precipitated with polyethylene glycol. To determine whether the altered binding observed with antibody 960 was due to increased affinity of the receptor for insulin or appearance of more insulin binding sites, binding studies were performed over a wide range of insulin concentrations. Analysis of the resulting binding curves indicated that 960 increased the affinity of the receptor for insulin 3 fold over control (b= 0.3 nM for 960, and 0.9 nM for 83-7, respectively). The antibody 960 also specifically increased insulin binding to intact, saponin-permeabilized IM-9 cell membranes. These results indicate that binding of 960 antibody to the juxtamembrane region of the insulin receptor alters the affmity of the receptor for insulin. Since tyrosine 960 in the juxtamembrane region has been suggested to play a role in receptor signalling, changes in receptor conformation in this region that are likely to account for the change in affinity may play a role in signal transduction.     

Solid Supported Vesicles for Bactericide Delivery

Abstract

Cationic and anionic liposomes have been prepared by extrusion from dipalmitoylphosphatidylcholine (DPPC) and its mixtures with cholesterol and dimethyldioctadecyltrimethylammonium bromide (DDAB) and with phosphatidylinositol (PI) respectively covering a range of composition from 0 to 19 mole % DDAB and PI. The adsorption of liposomal lipid from the liposome dispersion onto particles of silica and titanium dioxide in suspension has been studied as a function of liposome composition and concentration. The adsorption isotherms have been fitted using a Langmuir equation from which the binding constants and maximum surface coverage were obtained. The Gibbs energies of adsorption for the cationic liposomes were on average -61.0 ± 2.1 kJ mol^?1 (on silica) and -50.6 ± 2.9 kJ mol^?1 (on titanium dioxide). On average saturation adsorption is equivalent to 3 to 10 lipid monolayers on silica and 3 to 7 on titanium dioxide. Using liposomes encapsulating D-glucose it is demonstrated that there is almost no release of glucose on adsorption of the lipid, indicating that the liposomes are adsorbed intact to form a liposome monolayer on the particle surfaces. Adsorption of intact liposomes to form a close-packed liposome monolayer of solid supported vesicles (SSV) is shown to be equivalent to on average 7.0 ± 0.2 phospholipid monolayers. The SSVs are shown to have increased stability to disruption by surfactants and when carrying the oil-soluble bactericide, Triclosan?, to be capable of inhibiting the growth of oral bacteria from immobilised biofilms.     

Insulin binding in human skeletal muscle

Insulin binding to crude plasma membranes derived from human skeletal muscle was characterized. Incubations were performed for 22 h at 4°C. Typical insulin binding characteristics were found, i.e., (a) specificity for insulin, (b) pH sensitivity, (c) dissociation of insulin by the addition of excess insulin and (d) concave Scatchard curves. Half-maximal inhibition of ¹²⁵I-labeled-insulin binding occurred at 1 · 10^?8 M. Affinity constants were 0.76 · 10⁹ and 0.02 · 10⁹ M^?1 for the high- and low-affinity receptor (2-site model), respectively, and the corresponding receptor numbers were 89 and 1450 fmol/mg protein, respectively. The procedures employed permit the determination of insulin binding to small quantities of human muscle (approx. 250 mg).     

Insulin inhibition of calcium binding by human placental membrane

Insulin and its analogues displaced membrane-bound calcium within a physiological range of insulin concentration, in proportion to both biological potency and ability to displace porcine ¹²⁵I-labelled insulin from the insulin receptor. Mild tryptic digestion of the membrane reduced insulin binding but did not reduce specific calcium binding. Displacement of membrane-bound calcium by insulin was dependent on insulin binding to its intact receptor. These studies suggest that Ca²⁺ may exert a controlling influence on insulin-receptor binding in vivo.     

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.     

Ethnic Differences in the Responsiveness of Adipocyte Lipolytic Activity to Insulin

Objective: The goal of this study was to quantify differences in lipid metabolism and insulin sensitivity in black and white subjects to explain ethnic clinicopathological differences in type 2 diabetes. Research Methods and Procedures: The in vitro lipolytic activity of adipocytes isolated from obese black and white women was measured in the presence of insulin and isoproterenol. Insulin resistance was assessed in vivo using the euglycemic hyperinsulinemic clamp technique. Results: Fasting plasma levels of insulin and nonesterified fatty acid (NEFA) in black and white women were 67 ± 5 pM vs. 152 ± 20 pM (p < 0.01) and 863 ± 93 μM vs. 412 ± 34 μM (p < 0.01), respectively. Euglycemic hyperinsulinemic clamp studies showed that obese black subjects were more insulin‐resistant than their white counterparts (glucose infusion rates: 1.3 ± 0.2 vs. 2.2 ± 0.3 mg/kg per min; p < 0.05). Isolated adipocytes from white women were more responsive to insulin than those from black women with 0.7 nM insulin causing a 55 ± 4% inhibition of isoproterenol‐stimulated lipolysis compared with 27 ± 10% in black women (p < 0.05). Discussion: The low responsiveness of adipocyte lipolytic activity to insulin in black women in the presence of a relative insulinopenia may account for the high plasma NEFA levels seen in these women, which may, in turn, account for their higher in vivo insulin resistance. High NEFA levels may also contribute to the low insulin secretory activity observed in the obese black females. These data suggest that the pathogenesis of insulin resistance and type 2 diabetes within the black obese community is strongly influenced by their adipocyte metabolism.     

Chicken Insulin Discriminates Between Receptors for Insulin and Insulin-Like Growth Factor I on Centrally-and Peripherally-Derived Glial Cells

Abstract

Insulin and IGF-I receptors in G26–20 cells, derived from a mouse oligodendroglioma, and in RN-2 cells, derived from a rat Schwannoma, were characterized by specific binding to [¹²⁵I]insulin and [¹²⁵I]IGF-I respectively. In both cell lines, the Kd for insulin was 1.5 nM. Insulin receptor number was 33,000/cell for RN-2 cells and 17,000 receptors/ cell for G26–20 cells. RN-2 cells have 700,000 IGF-I receptors/cell with a Kd of 2 nM while G26–20 cells have 60,000 receptors/cell with an affinity of 4.9 nM. However, the independence of these two receptor populations in each cell type was equivocal since the subunit structure of these receptors appears identical by electrophoresis. In both cell lines, competition with insulin analogs for [¹²⁵I]insulin binding demonstrated chicken insulin>insulin>IGF-I. Competition for [¹²⁵I]IGF-I binding showed that IGF-I was approximately 85-fold more potent than insulin. Chicken insulin was ineffective at all concentrations. Thus, chicken insulin can be used as a specific ligand to unequivocally discriminate between IGF-I and insulin receptors and effects.     

Expression of Caveolin 1 Is Enhanced by DNA Demethylation during Adipocyte Differentiation. Status of Insulin Signaling

Caveolin 1 (Cav-1) is an essential constituent of adipocyte caveolae which binds the beta subunit of the insulin receptor (IR) and is implicated in the regulation of insulin signaling. We have found that, during adipocyte differentiation of 3T3-L1 cells the promoter, exon 1 and first intron of the Cav-1 gene undergo a demethylation process that is accompanied by a strong induction of Cav-1 expression, indicating that epigenetic mechanisms must have a pivotal role in this differentiation process. Furthermore, IR, PKB-Akt and Glut-4 expression are also increased during the differentiation process suggesting a coordinated regulation with Cav-1. Activation of Cav-1 protein by phosphorylation arises during the differentiation process, yet in fully mature adipocytes insulin is no longer able to significantly increase Cav-1 phosphorylation. However, these long-term differentiated cells are still able to respond adequately to insulin, increasing IR and PKB-Akt phosphorylation and glucose uptake. The activation of Cav-1 during the adipocyte differentiation process could facilitate the maintenance of insulin sensitivity by these fully mature adipocytes isolated from additional external stimuli. However, under the influence of physiological conditions associated to obesity, such as chronic inflammation and hypoxia, insulin sensitivity would finally be compromised.     

Insulin degradation by adipose tissue. Studies at several levels of cellular organization.

A systematic study of the degradation of physiological concentrations of 125I-labelled insulin was performed in intact fat-pads, isolated adipocytes and subcellular fractions of isolated adipocytes. The findings indicate that insulin is rapidly degraded to low-molecular-weight peptides and/or amino acids by the intact tissue and isolated cells. Of the total insulin-degradation products present after incubation with an intact fat-pad, 94% is recovered in the medium, indicating that these products are not retained by the cells or tissue. The plasma membranes do not degrade insulin significantly in the absence of reduced glutathione, and over 99% of the cellular degradative capacity is found in the postmicrosomal supernatant (cytosol). The cytosol degrades insulin to several labelled fragments that are intermediate in size between insulin and insulin A chain, as well as to the low-molecular-weight tissue degradation products. Inclusion of plasma membranes with cytosol accelerates the cleavage of the intermediate fragments to the size of the small products seen with the intact tissue. However, plasma membranes do not increase the initial step in the degradation of insulin when incubated with cytosol, suggesting that the insulin receptor is not involved with the direct cleavage of insulin. This study supports the hypothesis that the bulk of insulin degradation occurs in the adipocyte cytosol, where intermediate-sized fragments are generated and rapidly cleaved to smaller products by the plasma membrane and quickly released into the surrounding medium.     

Binding of sodium n-dodecyl sulphate and protons to catalase

The binding of sodium n-dodecyl sulphate to catalase has been measured by equilibrium dialysis in the pH range 3.2 to 10.0. On the acid side of the isoelectric point (pH 5.4) the surfactant anions initially bind to cationic sites on the protein and subsequent binding is cooperative. At high pH on the alkaline side of the isoelectric point only cooperative binding is observed. The binding data have been combined with protein titration curves to calculate the Gibbs energies of formation of protein titration curves to calculate the Gibbs energies of formation of protein surfactant proton complexes. Contributions to the Gibbs energies of complex formation by surfactant and protein binding have been estimated. The average Gibbs energies of surfactant binding to specific cationic sites are ca. 28 kJ mol^?1 and for cooperative binding ca. 15 kJ mol^?1.     

Further Evidence for a Role of Carbohydrates in Insulin Binding : Studies in Lectin-Purified Receptors

Abstract

Purification of liver membrane insulin receptors on concanavalin A-and ricin I-lectin columns gave a 15-fold enrichment in the insulin binding capacity per milligramm of protein. Final receptor and protein recoveries were 53 and 3.8 % respectively. Lectin-purification increased the receptor affinity for insulin, as indicated by a left-ward shift in the binding competition curve and a steeper slope in the Scatchard plot. Lectin-purification increased the receptor sensitivity towards the glycosidic probes. The maximal effects of β-galactosidase, ricin I (galactose-binding lectin) and α-mannosidase were markedly amplified : 80, 90 and 60 % inhibition, versus 45, 40 and 15 % with particulate membranes. The limulus polyphemus (LPA) and wheat germ (WGA) agglutinins (sialic acid- and N-acetyl-glucosaminyl-binding lectins) became effective in modifying the insulin binding : 45 and 80 % inhibition, respectively. The effects were dose-dependent, reversed by the monosaccharide competitors (lectin effects) and unrelated to the state of receptor occupancy. These findings indicate that, within the hormone recognition area, peptide chains containing galactose, mannose and N-acetyl-glucosamine are strictly required for insulin-receptor interaction and suggest that change in the receptor affinity is related to the role of carbohydrate in insulin binding.     

Adipocyte Fatty Acid‐binding Protein as a Determinant of Insulin Sensitivity in Morbid‐obese Women

The aim of the study was to evaluate human plasma circulating levels of adipocyte fatty acid‐binding protein (A‐FABP) and its relationship with proinflammatory adipocytokines and insulin resistance in a severely obese cohort, before and 1 year after a surgical gastric bypass. Plasmatic levels of A‐FABP were measured in 77 morbid‐obese women before and 1 year after bariatric surgery. Anthropometrical parameters and body composition by bioelectrical impedance analysis were determined. Circulating levels of soluble tumor necrosis factor receptor 2 (sTNFR2), Interleukin 18 (IL‐18), adiponectin, and high‐sensitive C‐reactive protein (hsCRP) were also analyzed. Insulin resistance by homeostasis model assessment of insulin resistance (HOMA‐IR) index was calculated. After massive weight loss, A‐FABP plasmatic levels decreased significantly [7.6 (8.9) vs. 4.3 (5.1); P < 0,001] but no association with circulating adipokines or proinflammatory cytokines, both at the beginning and at the end of follow‐up, was observed. A decrease in sTNFR2, IL‐18, hsCRP, and an increase in adiponectin levels (P < 0.001 in all cases) were observed after the gastric bypass. HOMA‐IR index improved 1 year after surgery and after multiple regression analysis remained associated with A‐FABP after controlling for confounding variables (β = 0.322, P = 0.014; R² for the model 0.281). In morbid‐obese women, plasma A‐FABP concentrations were dramatically reduced after gastric bypass surgery. After weight loss this protein contributed to HOMA‐IR index independently of proinflammatory/antinflammatory cytokine profile. Further studies are warranted to elucidate the role of A‐FABP in the pathogenesis of insulin resistance in morbid obesity.     

The hormonal regulation and interaction of concanavalin A and insulin binding in R3230AC mammary adenocarcinoma cells

To investigate the effects of concanavalin A on insulin binding to R323AC mammary carcinomas, initial experiments were performed to characterize binding of concanavalin A. Concanavalin A binding was found to be specific and saturable. Equilibrium binding experiments demonstrated that addition of low concentration of concanavalin A enhanced the binding of [³H]concanavalin A, suggestive of positively cooperative interactions. Binding of concanavalin A was responsive to hormonal alterations; tumor cells from diabetic rats showed enhanced binding of concanavalin A and insulin compared to cells from intact rats and administration of insulin to diabetic rats returned concanavalin A and insulin binding to levels seen in controls. Incubation of tumor cells with concanavalin A prior to addition of ¹²⁵I-labelled insulin resulted in a reduction of insulin-binding capacity; succinyl-concanavalin A did not affect binding of insulin. The percent inhibition of insulin binding by concanavalin A was highest at the lower insulin concentrations, providing a linearized Scatchard plot that yielded a calculated K_d value comparable to the low-affinity portion of the curvilinear Scatchard plot for insulin binding. The dissociation rate of bound insulin depended on receptor occupancy. Addition of concanavalin A after insulin binding reached equilibrium resulted in increased insulin binding hormone concentrations, decreased rates of dissociation of insulin and a loss of the correlation between receptor occupancy and dissociation rates. Concanavalin A alone demonstrated an insulin-like effect on glucose transport, which in these tumor cells represents a decrease in transport of 3-O-methylglucose. These suggest that binding of both concanavalin A and insulin to cells from this hormonally responsive neoplasm is under insulin regulation and demonstrates similar characteristics to those reported for a variety of normal cells. Furthermore, the interaction between concanavalin A and the cell membranes affects the affinity of the insulin receptor for insulin and appears to decrease the observed negative cooperativity.     

Lipid-Overloaded Enlarged Adipocytes Provoke Insulin Resistance Independent of Inflammation

In obesity, adipocyte hypertrophy and proinflammatory responses are closely associated with the development of insulin resistance in adipose tissue. However, it is largely unknown whether adipocyte hypertrophy per se might be sufficient to provoke insulin resistance in obese adipose tissue. Here, we demonstrate that lipid-overloaded hypertrophic adipocytes are insulin resistant independent of adipocyte inflammation. Treatment with saturated or monounsaturated fatty acids resulted in adipocyte hypertrophy, but proinflammatory responses were observed only in adipocytes treated with saturated fatty acids. Regardless of adipocyte inflammation, hypertrophic adipocytes with large and unilocular lipid droplets exhibited impaired insulin-dependent glucose uptake, associated with defects in GLUT4 trafficking to the plasma membrane. Moreover, Toll-like receptor 4 mutant mice (C3H/HeJ) with high-fat-diet-induced obesity were not protected against insulin resistance, although they were resistant to adipose tissue inflammation. Together, our in vitro and in vivo data suggest that adipocyte hypertrophy alone may be crucial in causing insulin resistance in obesity.     

Insulin stimulates GDP release from G proteins in the rat and human liver plasma membranes

Plasma membranes (1–2 mg protein) prepared from the livers of adult male rats and human organ donors were incubated with 0.6 μM [α-³²P] guanosine triphosphate (GTP) in an adenosine triphosphate (ATP)-regenerating buffer at 37°C for 1 h; during this incubation, the [³²P]GTP is hydrolyzed and the nucleotide that is predominantly bound to the membranes is [³²P] guanosine diphosphate (GDP). [³²P]GDP release from the liver membranes was proportional to the protein concentration and increased as a function of time. At 5 mM, Ca²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ maximally inhibited GDP release by 80–90%, whereas, 5 mM Cu²⁺ maximally stimulated the reaction by 100%. Therefore, cations were not included in the buffer used in the GDP release step. One μM Gpp(NH)p (5′-guanylylimidodiphosphate), a nonhydrolyzable analog of GTP, maximally stimulated [³²P]GDP release in the liver membranes by up to 30%. Although 10 nM Gpp(NH)p had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP/GTP exchange. In the rat membranes, 1–100 nM glucagon (used as a positive control) stimulated [³²P]GDP release by about 17% (P < .05); similarly, 0.1–100 nM insulin stimulated [³²P]GDP release by 10–13% (P < .05). In the human membranes, 10 pM to 100 nM insulin stimulated [³²P]GDP release by 7–10%. In the rat membranes, 10 nM insulin stimulated [³²P]GDP release by 17 and 24% at 2 and 4 min, respectively (P < .05); in the human membranes, 10 nM insulin stimulated [³²P]GDP release by about 9% at 2 and 4 min. Normal rabbit IgG (used as a control for insulin receptor antibody) by itself stimulated the GDP release by rat and human membranes. However, the stimulation of the GDP release by insulin receptor antibody was consistently higher than that observed with normal rabbit IgG. Four to 15 μg of insulin receptor antibody stimulated [³²P]GDP release by 12–22% (P < .05) and 7–14% in rat and human membranes, respectively. These results indicate that ligand binding to the insulin receptor results in a functional interaction of the receptor with a guanine nucleotide-binding transducer protein (G protein) and activation of GTP/GDP exchange.     

Insulin receptor kinase activity in rat adipocytes is decreased during aging

The tyrosine kinase activity of the insulin receptor derived from rat adipocyte plasma membranes was examined during aging. In the absence of insulin, autophosphorylation and histone H2B phosphorylation activities, measured with equal numbers of insulin receptors, were comparable among 3- and 24-month-old rats. In contrast, insulin-stimulated kinase activity was significantly reduced in the old animals. We have also found that the insulin dependent phosphorylation of a putative endogenous substrate of 60 kDa was drastically reduced in old animals. These results suggest that the decrease in kinase activity in old rats could be related with the insulin resistance of aging.     

Insulin binding to cells of several tissues of the early chick embryo.

The binding of I¹²⁵-labeled insulin to isolated cells from several tissues of the 3- and 4-day chick embryo was determined over a concentration range of insulin from 2 × 10^?11 to 2 × 10^?7M. The cells were obtained from limb bud and nonlimb bud tissues of the 4-day chick, from the headless 3-day chick embryo, and from cartilage of the 12-day embryo. The amount of bound insulin was found to be similar for the cells from the different embryonic tissues. Some implications of these findings for the interpretation of the nature of the binding sites and the teratogenic effect of insulin are discussed.