Michael F. Shanahan

Interactions of insulin, catecholamines and adenosine in the regulation of glucose transport in isolated rat cardiac myocytes

The regulation of the glucose transport system by catecholamines and insulin has been studied in isolated rat cardiomyocytes. In the basal state, 1-isoproterenol exhibited a biphasic concentration-dependent regulation of 3-O-methylglucose transport. At low concentrations (less than 10 nM), isoproterenol induced a maximal inhibition of 65-70% of the basal rates, while at higher concentrations (greater than 10 nM) a 25-70% stimulation of transport was observed. In the presence of adenosine deaminase, the inhibition of isoproterenol at low doses was attenuated. No effect of adenosine deaminase was observed on the stimulation of transport at high doses of isoproterenol. The inhibitory effect of isoproterenol returned when N6-phenylisopropyladenosine (a non-metabolizable analog of adenosine) was included along with adenosine deaminase. Dibutyryl cAMP and forskolin both inhibited basal transport rates. In the presence of maximally stimulating concentrations of insulin, cardiomyocyte 3-O-methylglucose transport was generally elevated 200-300% above basal levels. In the presence of isoproterenol, insulin stimulation was inhibited at both high and low concentrations of catecholamine, with maximum inhibition occurring at the lowest concentrations tested. When cells were incubated with both adenosine deaminase and isoproterenol, the inhibition of the insulin response was greater at all concentrations of catecholamine and was almost completely blocked at isoproterenol concentrations of 10 nM or less. Dibutyryl cAMP inhibited the insulin response to within 10% of basal transport levels, while forskolin completely inhibited all transport activity in the presence of insulin. These results suggest that catecholamines regulate basal and insulin-stimulated glucose transport via both cAMP-dependent and cAMP-independent mechanisms and that this regulation is modulated in the presence of extracellular adenosine.

Substitution of conserved tyrosine residues in helix 4 (Y143) and 7 (Y293) affects the activity, but not IAPS-forskolin binding, of the glucose transporter GLUT4

Six tyrosine residues (Y28, Y143, Y292, Y293, Y308, Y4321) which are conserved in all mammalian glucose transporters were substituted for phenylalanine by site‐directed mutagenesis, and mutant glucose transporters were transiently expressed in COS‐7 cells. Glucose transport activity as assessed by reconstitution of the solubilized transporters into lecithin liposomes was reduced by 70% in the mutant Y143F and appeared to be abolished in Y293F, but was not affected by substitution of Y28, Y292, Y308 and Y432. In contrast, covalent binding of the photolabel 125IAPS‐forskolin was normal in all mutants. Stable expression of the mutants Y143F, Y293F, and Y292F in LTK cells yielded identical results. These data indicate that only two of the 6 conserved helical tyrosines residues, located in helices 4 and 7, are essential for full activity, but not for IAPS‐forskolin binding of the GLUT4.

Polyphosphoinositide inclusion in artificial lipid bilayer vesicles promotes divalent cation-dependent membrane fusion.

Recent studies suggest that phosphoinositide kinases may participate in intracellular trafficking or exocytotic events. Because both of these events ultimately require fusion of biological membranes, the susceptibility of membranes containing polyphosphoinositides (PPIs) to divalent cation-induced fusion was investigated. Results of these investigations indicated that artificial liposomes containing PPI or phosphatidic acid required lower Ca2+ concentrations for induction of membrane fusion than similar vesicles containing phosphatidylserine, phosphatidylinositol, or phosphatidylcholine. This trend was first observed in liposomes composed solely of one type of phospholipid. In addition, however, liposomes designed to mimic the phospholipid composition of the endofacial leaflet of plasma membranes (i.e., liposomes composed of combinations of PPI, phosphatidylethanolamine, and phosphatidylcholine) also required lower Ca2+ concentrations for induction of aggregation and fusion. Liposomes containing PPI and phosphatidic acid also had increased sensitivity to Mg(2+)-induced fusion, an observation that is particularly intriguing given the intracellular concentration of Mg2+ ions. Moreover, the fusogenic effects of Ca2+ and Mg2+ were additive in vesicles containing phosphatidylinositol bisphosphate. These data suggest that enzymatic modification of the PPI content of intracellular membranes could be an important mechanism of fusion regulation.

Differential Labeling of Components in Human Erythrocyte Membranes Associated with the Transport of Glucose

The irreversible inhibition of glucose transport by 1-fluoro-2,4-dinitrobenzene (FDNB) has been used to identify membrane proteins possibly associated with glucose transport in human erythrocytes. D-Glucose was shown to enhance significantly the rate of FDNB inhibition of transport when present during the reaction, whereas cytochalasin B (CB) and D-maltose retarded this FDNB inhibition of transport. This modulation of the inhibition reaction formed basis for a double isotopic differential labeling technique using [14C]- and [3H] FDNB followed by SDS-polyacrylamide gel electrophoresis to distinguish transport-associated polypeptides from bulk membrane dinitrophenylated proteins. Reactions in the presence of CB or maltose revealed the presence of a differentially labeled polypeptide(s), with a molecular weight of approximately 60,000-65,000 daltons. This effect could in part be reversed in the presence of D-glucose but not L-glucose. Reactions in the presence of D-glucose resulted in two regions of differential labeling. One region was around 200,000 daltons and the other corresponded to a 90,000-dalton band. Extraction of membrane proteins with p-chloromercuribenzene sulfonate resulted in no loss of the 60,000-dalton peak, indicating that this labeled polypeptide(s) was firmly anchored in the hydrophobic core of the membrane. These results indicate that as many as three membrane polypeptides are differentially labeled by FDNB under conditions strongly associated with the inhibition of the glucose transport system and may be involved in the regulation of glucose transport.

Mutation of two conserved arginine residues in the glucose transporter GLUT4 supresses transport activity, but not glucose-inhibitable binding of inhibitory ligands

Two arginine residues (RR333/334) in the conserved GRR motif located in the endofacial loop between helix 8 and 9 of the glucose transporter GLUT4 were substituted for leucine and alanine, respectively. Reconstituted glucose transport activity of the construct (GLUT4-RR333/4LA) expressed in COS-7 or LM(TK-) cells was less than 10% of that of the wild-type GLUT4. In contrast, binding of the inhibitory ligand cytochalasin B and glucose-inhibitable photolabeling with IAPS-forskolin were not significantly affected. Exchange of a histidine residue (H337Q) previously believed to be involved in the binding of inhibitory ligands failed to affect any of the investigated parameters. These data suggest that positive charges in the GRR motif at the cytoplasmic surface of the transporter participate in the conformational changes of the carrier protein during the process of facilitated diffusion.

Localization of the binding domain of the inhibitory ligand forskolin in the glucose transporter GLUT-4 by photolabeling, proteolytic cleavage and a site-specific antiserum.

The binding domain of forskolin in the adipocyte/muscle-type glucose transporter (GLUT-4) was localized with the aid of the photoreactive derivative, [125I]IAPS-forskolin (3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin). Plasma membranes from insulin-treated rat adipocytes containing predominantly the GLUT-4 isoform were irradiated with UV light in the presence of [125I]IAPS-forskolin. The covalently labeled glucose transporters were isolated by immunoprecipitation with specific antiserum and partially digested with trypsin and elastase. The fragments were separated by gel electrophoresis, transferred on to nitrocellulose membranes, and identified by direct autoradiography and by immunoassay with antiserum against a peptide sequence corresponding to the C-terminus of GLUT-4. Digestion with a high-purity grade trypsin generated two photolabeled fragments with apparent molecular weights of 21 and 16 kDa. Since the antiserum detected two fragments with identical electrophoretic mobility, both labeled fragments appeared to contain the intact C-terminus of GLUT-4. In contrast, digestion with elastase generated only one photolabeled fragment with intact C-terminus at 21 kDa, and a smaller unlabeled fragment with intact C-terminus at 15 kDa. A less pure trypsin preparation generated two labeled (21 and 17 kDa) and one unlabeled (15 kDa) fragment with intact C-terminus. These data suggest that the site of covalent binding of IAPS-forskolin in the GLUT-4 is located within a region of 1-6 kDa defined by the difference between the unlabeled C-terminal fragment (15 kDa) and the labeled fragments (21, 17 and 16 kDa). Based on a tentative allocation of the fragments to the sequence of the GLUT-4, it is suggested that the covalent binding site of IAPS-forskolin is located between the membrane spanning helices 7-9, possibly in the proximity of helix 9.

Incorporating Nutrition into the Curriculum of a Midwestern Medical School

Nutrition has been identified as an area of need in medical school education by the American Society for Clinical Nutrition (ASCN), the American Medical Student Association, and the National Research Academy of Sciences.1-3 In fact, the American Medical Association (AMA) made specific recommendations for increasing the development of medical nutrition education curricula in the Medical Society Recommendations on Nutrition Education for Medical Schools.4 Along with other evidence, the report recognized that the Association of American Medical Colleges (AAMC) Medical School Graduation Questionnaire data show that almost 2 of 3 fourth-year medical students believe that the time devoted to nutrition in medical school has been inadequate. The purpose of this project was to expand and strengthen nutrition in the curriculum of a Midwest university medical school. The specific objectives of this project were to integrate nutr ition into the medical curriculum by developing nutritionbased resource sessions and minicases and to increase the quantity of nutrition education resources. In the late 1990s, the 4-year undergraduate medical school curriculum of this Midwest university was redesigned (Table 1), creating Curriculum 2000.5 Curriculum 2000 provided an overall focus on clinical case–based, studentdirected learning in a small-group setting through problem-based learning. Prior to Curriculum 2000, nutrition education was not part of the curriculum; thus, this team was charged with expanding the basic sciences to include nutrition.This challenge, along with a grant received from the Illinois Attorney General’s Office, led to an initiative to incorporate nutrition into Curriculum 2000. Initially, the research team (including a nutritionist, a physiologist, a biochemist, an anatomist, and clinicians) reviewed the curriculum to determine current material delivered to students and placement of nutrition education to minimize disruption of the existing curriculum structure. Members of the research team also participated in the National Board of Medical Examiners Faculty Item Review Program, allowing them to compare the nutritionbased content of the examination with the nutrition content placed in the curriculum. Based on the gaps identified and recommendations from the AMA,ASCN, and AAMC, the following initiatives were taken.1-5

GLUCOSE TRANSPORT ACTIVITY AND LIGAND BINDING (CYTOCHALASIN B, IAPS-FORSKOLIN) OF CHIMERIC CONSTRUCTS OF GLUT2 AND GLUT4 EXPRESSED IN COS-7-CELLS

Chimeric constructs of glucose transporters GLUT2 and GLUT4 were transiently expressed in COS-7 cells in order to determine regions of the proteins responsible for their differences in activity and ligand binding. Exchange of the C-terminal tail (aa 479-509) of GLUT4 failed to affect glucose transport activity assayed at 1 mM glucose or ligand binding (cytochalasin B, IAPS-forskolin). In contrast, exchange of the C-terminal half of GLUT4 (aa 222-509) for that of GLUT2 markedly reduced ligand binding (Kd of cytochalasin B binding 1.88 +/- 0.2 microM vs. 0.21 +/- 0.06 in the wild-type GLUT4), and moderately (25%) reduced glucose transport activity. These data support the conclusion that the domains determining differences in ligand binding between GLUT4 and GLUT2 are located in the C-terminal half of the glucose transporters.

Selective extraction of an intrinsic fat-cell plasma-membrane glycoprotein by Triton X-100. Correlation with [3H]cytochalasin B binding activity.

Cytochalasin B is an extraordinarily potent inhibitor of glucose-transport systems which operate by facilitated diffusion in a variety of mammalian cell types studied [l-lo]. In red cells, most of the high-affinity binding of cytochalasin B can be inhibited by addition of unlabelled glucose, indicating competition for similar binding sites [ 111. In fat cells [lo] and fibroblasts [ 121 kinetics characteristic of competitive interaction between cytochalasin B and glucose are not observed, although binding of [3H]cytochalasin B to high affinity sites does parallel transport inhibition [lo]. These findings have suggested that high affinity cytochalasin B-binding sites may be related to one or more protein components of the plasma-membrane hexose-transport system in a given cell type. We have recently found [13] that all of the fat-cell plasma-membrane proteins, except the 94 000 and 78 000 dalton glycoproteins and a minor 56 000 dalton fraction, are eluted from the membrane upon treatment with dimethylmaleic anhydride (DMMA) as described for erythrocyte membranes [ 14,151. This extracted membrane preparation was shown to retain stereospecific hexose-transport activity which was sensitive to inhibition by cytochalasin B. Similarly, [3H]cytochalasin B binding to this membrane exhibited a dissociation constant identical to that observed with intact fat-cell plasma membranes. The present report describes our development of methodology designed to further differentially elute protein