Post-binding events in insulin action.
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Adipose Tissue↗
Biomedical subjects
R Horuk
Publications and source records attributed to R Horuk.
Photoaffinity labeling of the glucagon receptor with a new glucagon analog.
The preparation, purification and characterization of N epsilon-4- azidophenylamidinoglucagon are described. This photoreactive peptide was found to be 50% as potent as native glucagon in competing with 125I-labeled glucagon for binding to glucagon receptors on rat liver plasma membranes. Similarly, the analog was 50% as potent as native glucagon in its ability to stimulate adenylate cyclase. The photoreactive glucagon analog was radioiodinated to high specific activity with iodine-125 and was used to label rat liver plasma membrane proteins. Analysis of labeled membrane proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed covalent incorporation predominantly into a protein of relative molecular mass, Mr, of 50 000-60 000. Occasionally a protein of Mr 170 000-180 000 was also labeled. Irradiation of membranes in the presence of unlabeled glucagon or GTP selectively inhibited the labeling of the 50 000-60 000-Mr protein(s). As a result of these studies we suggest that the sodium-dodecyl-sulfate-dissociated glucagon receptor is a 50 000-60 000-Mr protein.
Adenylyl Cyclases↗
Purification of the photoaffinity-labeled glucagon receptor by gel electrophoretic methods.
Rat liver plasma membrane glucagon receptor has been purified with a yield of 0.01% to an estimated homogeneity of 32-60%, using a 2-stage electrophoretic procedure. SDS-solubilized membrane proteins labeled by the photoaffinity-agent, Ne-4-azidophenylamidinoglucagon (APA-glucagon), were separated by polyacrylamide gel electrophoresis in SDS-containing buffers. Gel slices corresponding to the molecular weight of the receptor were excised, electrophoretically extracted and concentrated. The concentrate was subjected to isoelectric focusing on Sephadex to yield a purified product in which the photoaffinity-labeled receptor, with a molecular weight of 56K and a pI' of 5.9, is the sole major component.
Affinity Labels↗
Identification and characterization of the rat adipocyte glucose transporter by photoaffinity crosslinking.
The photoaffinity crosslinking agent hydroxysuccinimidyl-4-azidobenzoate has been used to attach [3H]cytochalasin B to a rat adipocyte low-density microsomal membrane protein of 45-50 kDa. The characteristics of the [3H]cytochalasin B-labeled protein are consistent with those of the adipocyte glucose transporter. The low-density microsomes from cells incubated without insulin incorporate twice the amount of radioactivity per mg membrane protein than low-density microsomes derived from insulin-stimulated cells. This value agrees with the distribution of glucose transporters measured in this intracellular membrane fraction prepared from basal and insulin-treated cells by [3H]cytochalasin B binding. Preincubation of membranes with 500 mM D-glucose reduces the photoaffinity crosslinking by 48% relative to that observed with 500 mM L-glucose. Isoelectric focusing of low-density microsomes containing the photoaffinity crosslinked transporter yields three bands of radioactivity focusing at pH values of 5.5, 4.5, and 4.2 respectively. Following isolation from the isoelectric focusing gel and SDS-polyacrylamide gel electrophoresis, all three peaks can be shown to contain a band of 45-50 kDa which crossreacts with an antiserum raised against the purified human erythrocyte glucose transporter. These results suggest that the identification, isolation and purification of the adipocyte glucose transporter is now possible using the techniques described above.
Adipose Tissue↗
Proposed mechanism of insulin-resistant glucose transport in the isolated guinea pig adipocyte. Small intracellular pool of glucose transporters.
A marked resistance to the stimulatory action of insulin on glucose metabolism has previously been shown in guinea pig, compared to rat, adipose tissue and isolated adipocytes. The mechanism of insulin resistance in isolated guinea pig adipocytes has, therefore, been examined by measuring 125I-insulin binding, the stimulatory effect of insulin on 3-0-methylglucose transport and on lipogenesis from [3-3H]glucose, the inhibitory effect of insulin on glucagon-stimulated glycerol release, and the translocation of glucose transporters in response to insulin. The translocation of glucose transporters was assessed by measuring the distribution of specific D-glucose-inhibitable [3H]cytochalasin B binding sites among the plasma, and high and low density microsomal membrane fractions prepared by differential centrifugation from basal and insulin-stimulated cells. At a glucose concentration (0.5 mM) where transport is thought to be rate-limiting for metabolism, insulin stimulates lipogenesis from 30 to 80 fmol/cell/90 min in guinea pig cells and from 25 to 380 fmol/cell/90 min in rat cells with half-maximal effects at approximately 100 pM in both cell types. Insulin similarly stimulates 3-O-methylglucose transport from 0.40 to 0.70 fmol/cell/min and from 0.24 to 3.60 fmol/cell/min in guinea pig and rat fat cells, respectively. Nevertheless, guinea pig cells bind more insulin per cell than rat cells, and insulin fully inhibits glucagon-stimulated glycerol release. In addition, the differences between guinea pig and rat cells in the stimulatory effect of insulin on lipogenesis and 3-O-methylglucose transport cannot be explained by the greater cell size of the former compared to the latter (0.18 and 0.09 micrograms of lipid/cell, respectively). However, the number of glucose transporters in the low density microsomal membrane fraction prepared from basal guinea pig cells is markedly reduced compared to that from rat fat cells (12 and 70 pmol/mg of membrane protein, respectively) and the translocation of intracellular glucose transporters to the plasma membrane fraction in response to insulin is correspondingly reduced. These results suggest that guinea pig adipocytes are markedly resistant to the stimulatory action of insulin on glucose transport and that this resistance is the consequence of a relative depletion in the number of intracellular glucose transporters.
Adipose Tissue↗
Partial purification and characterization of the glucagon receptor.
Specific labeling of liver plasma membrane glucagon receptors has been achieved by the photoincorporation of a 125I-labeled photoderivative of glucagon, NE-4-azidophenylamidinoglucagon. Identification of glucagon receptors was facilitated by irradiating membranes in the presence of excess unlabeled glucagon. Isoelectric focusing of radioiodinated membrane proteins revealed one major band of glucagon displaceable material which had an isoelectric point of 5.85. When this material was isolated and run on SDS-polyacrylamide gels a major labeled band of Mr55000 was obtained which had properties consistent with those of the glucagon receptor. These studies indicate that a purification of the glucagon receptor of greater than 700-fold can be attained through the use of isoelectric focusing and SDS-polyacrylamide electrophoresis.
Animals↗
A monomeric insulin from the casiragua: molecular model building using computer graphics.
Interactive computer graphics have been used to construct a three-dimensional model of casiragua insulin assuming a tertiary structural homology with that of porcine insulin. The hydrophobic core of the molecule is conserved, but the hydrophobic surfaces involved in formation of dimers and hexamers in porcine insulin are more hydrophilic and contain charged residues. These observations are consistent with a close similarity of the far ultra violet circular dichroism of casiragua insulin and porcine insulin, and with the inability of casiragua insulin to self-associate. The low receptor-binding potency appears to result mainly from the substitution of B26-tyrosine by an arginine.
Animals↗
A monomeric insulin from the porcupine (Hystrix cristata), an Old World hystricomorph.
The insulins of New World hystricomorph rodents exhibit many novel amino acid changes in primary structure when compared with other mammalian insulins. These changes give rise to unusual properties (low potency, failure to self-associate) not shared by other naturally-occurring insulins. We report here on the primary structure, zinc-binding properties and circular dichroism (CD) of porcupine-insulin (Hystrix cristata), the first Old World hystricomorph insulin to be investigated, and discuss the changes in primary structure of the hormone in relation to its properties. Residue B22 is strongly implicated as being responsible for the unusual properties of porcupine insulin.
Amino Acid Sequence↗
Evolutionary change in the insulin receptors of hystricomorph rodents.
INSULIN receptors have been thought to have remained unaltered despite evolutionary changes in the hormone(1). Because insulins from hystricomorph rodents are known to be highly substituted compared with other mammalian insulins, we decided to investigate the insulin receptor of some hystricomorphs to determine whether evolutionary change had occurred within the receptor itself. Here we present the first evidence that hystricomorph rodent insulin receptors have undergone evolutionary change.
Journal Article↗