Recombinant Sindbis virus as an expression system for cell biology.
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Animals↗
Biomedical subjects
R C Piper
Publications and source records attributed to R C Piper.
Insulin resistance, diabetes, and the insulin-regulated trafficking of GLUT-4.
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Adipocytes↗
Yeast Vps45p is a Sec1p-like protein required for the consumption of vacuole-targeted, post-Golgi transport vesicles.
Over 45 VPS genes (vacuolar protein sorting) in Saccharomyces cerevisiae are necessary for the correct sorting and delivery of vacuolar hydrolases. Yeast strains carrying mutations in a subset of these VPS genes (class D vps mutants) are also defective in the segregation of vacuolar material into the developing daughter cell and are morphologically characterized by having large central vacuoles. The class D VPS gene products, which include a Rab5 homologue (VPS21/YPT51) and a syntaxin homologue (PEP12/VPS6), have been proposed to function together at a particular step along the vacuolar protein sorting pathway. We have cloned another class D VPS gene, VPS45, which is homologous to a growing family of genes that encode Sec1p-like proteins. Vps45p is predicted to be a hydrophilic protein of 577 amino acids with a molecular mass of 67 kDa. Fractionation studies show that Vps45p is a peripheral membrane protein that cofractionates with Golgi-like membranes, consistent with Vps45p functioning in membrane traffic between the Golgi and the vacuole. Using a temperature-sensitive allele of VPS45, we show that inactivation of Vps45p causes the rapid accumulation of small (40-60 nm) vesicles and secretion of the vacuolar hydrolase carboxypeptidase Y. Because the entire yeast secretory pathway is functional after the temperature-induced inactivation of Vps45p, we conclude that the accumulated vesicles represent transport intermediates between the Golgi and the vacuole.
Amino Acid Sequence↗
GLUT4 phosphorylation and inhibition of glucose transport by dibutyryl cAMP.
To investigate the mechanism responsible for the inhibition of glucose transport by dibutyryl cAMP (Bt2cAMP), two different transporter isoforms (GLUT1 and GLUT4) and several GLUT1/4 chimeric transporters were expressed in Chinese hamster ovary (CHO) cells by using a Sindbis virus expression system. Bt2cAMP inhibited GLUT4-mediated 2-deoxy[3H]glucose (2DOG) uptake by 50% but was without effect on GLUT1-mediated uptake. When the subcellular distribution of GLUT4 was assessed by quantitative immunocytochemistry, neither the overall concentration of GLUT4 nor the regional distribution of GLUT-4 within the plasma membrane was found to be altered by Bt2cAMP. Thus, inhibition of 2DOG uptake by Bt2cAMP appears to be due to a decrease in transporter activity rather than a decrease in the number of transporters exposed at the plasma membrane. By using chimeric transporters, a region of GLUT4 necessary for the inhibitory effect of Bt2cAMP was localized to the last 29 amino acids in the COOH terminus. This intracellular region contains the site (Ser488) phosphorylated in vitro by cAMP-dependent protein kinase (cAdPK). Changing Ser488 to an Ala abolished phosphorylation of GLUT4; however, the inhibitory effect of Bt2cAMP on glucose transport was not diminished by this mutation. Therefore, phosphorylation of GLUT4 was not required for the inhibition. The effects of other nucleotides on GLUT4 transport activity were assessed to investigate the role of cAdPK. Uptake of 2DOG by GLUT4 was inhibited by 8-bromo-AMP, but not by 8-bromo-cAMP, suggesting that the inhibitory effect did not involve activation of cAdPK. Results consistent with this interpretation were obtained with CHO cells (line 10248), which express a cAdPK that is resistant to activation by cAMP. No difference in the concentrations of Bt2cAMP required to inhibit GLUT4-mediated transport was observed in normal CHO cells and 10248 cells. The results presented suggest that the inhibitory effects of Bt2cAMP could be mediated by direct binding of a nucleotide to GLUT4 at a site involving the intracellular COOH terminus of the transporter.
Animals↗
GLUT-4 NH2 terminus contains a phenylalanine-based targeting motif that regulates intracellular sequestration.
Expression of chimeras, composed of portions of two different glucose transporter isoforms (GLUT-1 and GLUT-4), in CHO cells had indicated that the cytoplasmic NH2 terminus of GLUT-4 contains important targeting information that mediates intracellular sequestration of this isoform (Piper, R. C., C. Tai, J. W. Slot, C. S. Hahn, C. M. Rice, H. Huang, D. E. James. 1992. J. Cell Biol. 117:729-743). In the present studies, the amino acid constituents of the GLUT-4 NH2-terminal targeting domain have been identified. GLUT-4 constructs containing NH2-terminal deletions or alanine substitutions within the NH2 terminus were expressed in CHO cells using a Sindbis virus expression system. Deletion of eight amino acids from the GLUT-4 NH2 terminus or substituting alanine for phenylalanine at position 5 in GLUT-4 resulted in a marked accumulation of the transporter at the plasma membrane. Mutations at other amino acids surrounding Phe5 also caused increased cell surface expression of GLUT-4 but not to the same extent as the Phe5 mutation. GLUT-4 was also localized to clathrin lattices and this colocalization was abolished when either the first 13 amino acids were deleted or when Phe5 was changed to alanine. To ascertain whether the targeting information within the GLUT-4 NH2-terminal targeting domain could function independently of the glucose transporter structure this domain was inserted into the cytoplasmic tail of the H1 subunit of the asialoglycoprotein receptor. H1 with the GLUT-4 NH2 terminus was predominantly localized to an intracellular compartment similar to GLUT-4 and was sequestered more from the cell surface than was the wild-type H1 protein. It is concluded that the NH2 terminus of GLUT-4 contains a phenylalanine-based targeting motif that mediates intracellular sequestration at least in part by facilitating interaction of the transporter with endocytic machinery located at the cell surface.
Amino Acid Sequence↗
Targeting of mammalian glucose transporters.
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Amino Acid Sequence↗
Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity.
This report compares the use of the lactate dehydrogenase (pLDH) assay with 3H-hypoxanthine incorporation and Giemsa microscopy for the evaluation of anti-malaria drug inhibition of the growth of P. falciparum in vitro. The inhibition profiles and IC50 determinations of the pLDH assay were directly comparable to those determined by the radioactive uptake and microscopic methods. Furthermore, the pLDH culture sensitivity assay is reproducible, easily interpreted, rapid and inexpensive to perform, suggesting field applicability.
Animals↗
GLUT4 facilitates insulin stimulation and cAMP-mediated inhibition of glucose transport.
The glucose transporter isoform GLUT4 is found only in cells that exhibit insulin-sensitive glucose transport. To investigate the function of this transporter, L6 myoblasts were stably transfected with GLUT4 cDNA. GLUT4 underwent insulin-dependent movement to the cell surface in myoblasts overexpressing the transporter. One cell line (243-6) expressed sufficient levels of the GLUT4 protein to study insulin-dependent glucose transport. Unlike wild-type L6 cells, 243-6 myoblasts exhibited two features that are characteristic of differentiated muscle fibers and adipocytes in vivo: a large insulin-stimulated component of glucose transport and inhibition of this stimulated component by cAMP. Relative to normal L6 cells, 243-6 cells responded to insulin or insulin-like growth factor 1 with a 5-fold larger increase in 2-deoxy[3H]glucose uptake. N6,O2'-Dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) did not inhibit transport in normal L6 myoblasts, which express only GLUT1, but inhibited IGF-1/insulin-stimulated transport by 50% in 243-6 cells. The effect of cAMP was investigated further by using Chinese hamster ovary cells transiently expressing GLUT1 and GLUT4. Bt2cAMP inhibited glucose transport only in Chinese hamster ovary cells expressing GLUT4. These results indicate that cAMP-mediated inhibition of glucose transport is dependent on expression of the GLUT4 isozyme.
Animals↗
The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus.
GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.
Amino Acid Sequence↗
Interaction of insulin and exercise on glucose transport in muscle.
Glucose transport is the rate-limiting step for glucose utilization in muscle. In muscle and adipose tissue, glucose transport is acutely regulated by such factors as insulin and exercise. Translocation of glucose transporters (GLUT4) from an intracellular domain to the cell surface is the major mechanism for this regulation. Using immunocytochemistry, the intracellular distribution of GLUT4 under resting conditions is similar in adipocytes and myocytes. GLUT4 is concentrated in tubulovesicular structures either in the trans-Golgi region or in the cytosol, often close to the cell surface but not on the cell surface. After stimulation, cell surface GLUT4 labeling is increased by as much as 40-fold. GLUT4 is chronically regulated by altered gene expression. Neural and/or contractile activity regulates GLUT4 expression in muscle: 1) GLUT4 levels differ among muscles of different fiber type; 2) GLUT4 levels in muscle are increased with exercise training and decreased with denervation; and 3) cultured muscle cells, which lack an intact nerve supply, express very low levels of GLUT4. GLUT4 expression appears to be regulated in parallel with many oxidative enzymes in muscle, suggesting that there may be a unified developmental program that determines the overall metabolic properties of a particular muscle. Preliminary evidence suggests that impaired GLUT4 expression in muscle is not the primary defect associated with insulin resistance. Nevertheless, it is conceivable that the adaptive increase in muscle GLUT4 that is found with exercise training may have beneficial effects in insulin-resistant states such as non-insulin-dependent diabetes.
Animals↗
Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type.
Insulin stimulates glucose transport in adipocytes via the rapid redistribution of the GLUT1 and GLUT4 glucose transporters from intracellular membrane compartments to the cell surface. Insulin sensitivity is dependent on the proper intracellular trafficking of the glucose transporters in the basal state. The bulk of insulin-sensitive transport in adipocytes appears to be due to the translocation of GLUT4, which is more efficiently sequestered inside the cell and is present in much greater abundance than GLUT1. The cell type and isoform specificity of GLUT4 intracellular targeting were investigated by examining the subcellular distribution of GLUT1 and GLUT4 in cell types that are refractory to the effect of insulin on glucose transport. Rat GLUT4 was expressed in 3T3-L1 fibroblasts and HepG2 hepatoma cells by DNA-mediated transfection. Transfected 3T3-L1 fibroblasts over-expressing human GLUT1 exhibited increased glucose transport, and laser confocal immunofluorescent imaging of GLUT1 in these cells indicated that the protein was concentrated in the plasma membrane. In contrast, 3T3-L1 fibroblasts expressing GLUT4 exhibited no increase in transport activity, and confocal imaging demonstrated that this protein was targeted almost exclusively to cytoplasmic compartments. 3T3-L1 fibroblasts expressing GLUT4 were unresponsive to insulin with respect to transport activity, and no change was observed in the subcellular distribution of the protein after insulin administration. Immunogold labeling of frozen ultrathin sections revealed that GLUT4 was concentrated in tubulo-vesicular elements of the trans-Golgi reticulum in these cells. Sucrose density gradient analysis of 3T3-L1 homogenates was consistent with the presence of GLUT1 and GLUT4 in discrete cytoplasmic compartments. Immunogold labeling of frozen thin sections of HepG2 cells indicated that endogenous GLUT1 was heavily concentrated in the plasma membrane. Sucrose density gradient analysis of homogenates of HepG2 cells expressing rat GLUT4 suggested that GLUT4 is targeted to an intracellular location in these cells. The density of the putative GLUT4-containing cytoplasmic membrane vesicles was very similar in HepG2 cells, 3T3-L1 fibroblasts, 3T3-L1 adipocytes, and rat adipocytes. These data indicate that the intracellular trafficking of GLUT4 is isoform specific. Additionally, these observations support the notion that GLUT4 is targeted to its proper intracellular locale even in cell types that do not exhibit insulin-responsive glucose transport, and suggest that the machinery that regulates the intracellular targeting of GLUT4 is distinct from the factors that regulate insulin-dependent recruitment to the cell surface.
Adipose Tissue↗
Differential sorting of two glucose transporters expressed in insulin-sensitive cells.
The insulin-regulatable glucose transporter (IRGT) is specifically expressed in muscle and fat cells and undergoes translocation from an intracellular compartment to the cell surface following acute insulin treatment. This study examined sorting differences between the IRGT and the homologous HepG2/erythrocyte/brain glucose transporter (HepG2 GT) when expressed together in insulin-responsive 3T3-L1 adipocytes. The ratio of the amount of transporter per unit protein in the plasma membrane fraction vs. the intracellular membrane fraction was 1:2 for the HepG2 GT and 1:30 for the IRGT. Insulin treatment increased the plasma membrane concentration of the IRGT by 10-fold and the HepG2 GT by 3.5-fold. This distribution was confirmed by confocal immunofluorescence microscopy. Differential sorting within intracellular organelles was evident by sucrose gradient analysis and immunoisolation of transporter vesicles and by double immunofluorescence labeling. We propose that differential sorting at an intracellular locus preferably withdraws the IRGT from a pathway which is in close communication with the plasma membrane, thus allowing the IRGT to regulate glucose entry into fat and muscle cells in a highly insulin-regulated fashion.
Adipose Tissue↗
Repeated transplacental transmission of Neospora caninum in dogs.
Four litters of German Shorthaired Pointers from one owner developed a toxoplasmosis-like illness. According to the records, 29 of 39 dogs had hind limb paralysis. Six dogs from 2 litters were necropsied and had generalized encephalomyelitis. Tachyzoites and tissue cysts of Neospora caninum were found in the brain and spinal cord of each dog. Lesions were found in the eyes, extraocular muscles, or both in all of the dogs, and N caninum was detected microscopically in the eyes (retina and choroid in 1 dog), extraocular muscles, or both in 5 of the 6 dogs. Ocular lesions consisted of focal retinitis, choroiditis, mild nonspecific iridocyclitis, and myositis of extraocular muscles. Organisms stained with anti-N caninum serum, but not with anti-Toxoplasma gondii serum in an immunohistochemical test, except in 1 dog. In one dog, aged thick-walled N caninum tissue cysts reacted mildly with anti-T gondii serum.
Animals↗
Hepatic changes produced by 30-day administration of a novel aminocyclitol antibiotic, trospectomycin sulfate, to laboratory animals.
The studies described here were done to characterize the hepatic response to a new aminocyclitol antibiotic, trospectomycin sulfate, administered intravenously (beagle dog) or subcutaneously (Sprague-Dawley rat) at a variety of dose levels, to investigate reversibility of observed changes, and to document any untoward effects of subchronic trospectomycin sulfate administration. Both species showed significant elevations in serum levels of alanine and aspartate transaminases in higher dose groups. In the dog only, a transient neuromuscular blockade was also observed within higher dose groups. No other functional, morphological, or serum chemical changes were observed. Examination of liver by electron microscopy revealed the presence of cytoplasmic lamellar inclusion bodies, concentrated in the bile canalicular region of the hepatocytes. Occurrence of the lamellar bodies and coincident transaminase increases were found to be reversible upon discontinuance of treatment (studied in the dog). Electron microscopy of acid phosphatase cytochemistry in the rat indicated that most, but not all, of the lamellar bodies contained this enzyme. This observation suggests that they may be derived from the lysosome, or once formed become lysosomal.
Acid Phosphatase↗
Differential effects of cefmetazole sodium on the reproductive system of infant and pubertal male rats.
The effects of cefmetazole (CMZ), a cephem antibiotic which contains the N-methyltetrazolethiol (NMTT) side-chain moiety, were compared in infant (6-42 days of age) and pubertal (6-10 weeks of age) male Sprague-Dawley rats. High doses of either CMZ or free NMTT caused reductions in testicular weight and delayed maturation of spermatogenic germ cells in the testes of infant rats, implicating NMTT as the active component in causing these effects. Pubertal rats expressed neither of these effects, even when treated with doses of CMZ far in excess of those used in infant rats. The effects of CMZ and NMTT on testicular weights and histologic features of testes of rats treated as infants were mainly reversed when these animals were examined 35 and 70 days after cessation of treatment. All reproductive functional parameters were normal in mating studies using male rats which had been treated with CMZ or NMTT as infants and allowed to recover. Because of the species differences in rates of sexual maturation and the greater rate at which rats metabolize CMZ to NMTT, the relevance to humans of the testicular effects of CMZ in infant rats is unknown.
Animals↗
Preclinical safety studies of cefmetazole.
In order to assess the safety of cefmetazole, preclinical multiple-dose parenteral studies, varying from one to three months in length, were conducted in Sprague-Dawley rats and beagle dogs. Although the largest doses used were in multiples of several times the weight-adjusted doses intended for humans, cefmetazole was generally well tolerated. The principal adverse effect noted in the adult rats receiving the largest doses (2000 and 2500 mg/kg/day) of cefmetazole was slight elevation of serum alanine aminotransferase. Infant rats injected subcutaneously with 300 mg/kg/day or more of cefmetazole for 35 consecutive days had reversible reductions in testicular weight and maturation of spermatogenesis, but not lasting discernible effect on reproductive function. The most consistent effects of longterm multiple dosing with cefmetazole in dogs consisted of vomiting and retching during dosing and reversible haematological changes (mild regenerative anaemia, positive Coombs' test, clinically-silent thrombocytopenia) in a number of the dogs. These findings supported the interpretation that cefmetazole was acceptably safe for clinical studies in humans.
Animals↗
Pathogenesis of cardiovascular alterations in dogs treated with minoxidil.
Minoxidil and other potent vasodilators cause coronary arterial injury, right atrial hemorrhagic lesions, and subendocardial necrosis in dogs. This paper discusses the pathogenesis of coronary arterial and right atrial lesions associated with minoxidil in the dog. Acute coronary vascular injury characterized by segmental medial hemorrhage and necrosis and perivascular inflammation occurred only during the first few days of treatment, after which tolerance to further acute injury developed. At 30 d or more of treatment, coronary vascular injury was characterized by perivascular fibrosis rarely attended by medial distortion or hyperplasia and subintimal thickening, changes consistent with responses to previous injury. Right atrial hemorrhagic lesions, unlike coronary vascular injury, often became progressively more extensive with continued treatment. At 3 d, atrial hemorrhage and inflammation were confined to the subepicardium of the right atrium, evidently around affected subepicardial branches of the right coronary artery. At 30 d, fibrovascular proliferative right atrial lesions (granulation tissue with evidence of continual hemorrhage) extended from the epicardium to the myocardium, with eventual replacement of the atrial wall by mature connective tissue at 1 yr of treatment. Minoxidil-induced cardiovascular lesions were not prevented by treatment with a beta-blocker (propranolol), or an alpha-blocker (dibenzylene), or by sympathetic neural activity suppression (surgical sympathectomy or constant carotid sinus nerve stimulation), suggesting that the sympathetic response to the pharmacologic activity of minoxidil was not responsible for the induction of the cardiovascular lesions. Minoxidil-related vascular lesions were confined to the most pharmacologically responsive segment of the arterial system, the coronary arteries, suggesting that medial injury may have been associated with tensile changes in the arterial wall.
Adrenergic beta-Antagonists↗
An enzyme immunoassay for the flow cytometer (FEIA).
This report describes the use of a fluorescent activated cell sorter (FACS) in combination with an enzyme immunosorbent assay (EIA). This combination of techniques expands the versatility of the flow cytometer. It introduces a new set of fluorescent enzyme products for antigen detection. These highly substantive fluorescent compounds permit the flow cytometer to quantify cell-EIA reactions and also to delineate subpopulations of cells with different quantities of surface antigens. Because the FEIA product is colored, as well as fluorescent, a simple light microscope may also be used to define the distribution of the label on the cell surface. These techniques have been applied to the examination of antigens on human erythrocytes, human T cell lymphoma cells (H9), and to surface markers on the tissue culture cell line K562.
Antigens↗