scholarly journals Nanoparticle Delivered Human Biliverdin Reductase-Based Peptide Increases Glucose Uptake by Activating IRK/Akt/GSK3 Axis: The Peptide Is Effective in the Cell and Wild-Type and Diabetic Ob/Ob Mice

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Peter E. M. Gibbs ◽  
Tihomir Miralem ◽  
Nicole Lerner-Marmarosh ◽  
Mahin D. Maines
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
Glucose Uptake ◽  
Animal Studies ◽  
Cultured Cells ◽  
Kinase Domain ◽  
Terminal Segment ◽  
Wild Type ◽  
Stimulation Of

Insulin’s stimulation of glucose uptake by binding to the IRK extracellular domain is compromised in diabetes. We have recently described an unprecedented approach to stimulating glucose uptake. KYCCSRK (P2) peptide, corresponding to the C-terminal segment of hBVR, was effective in binding to and inducing conformational change in the IRK intracellular kinase domain. Although myristoylated P2, made of L-amino acids, was effective in cell culture, its use for animal studies was unsuitable. We developed a peptidase-resistant formulation of the peptide that was efficient in both mice and cell culture systems. The peptide was constructed of D-amino acids, in reverse order, and blocked at both termini. Delivery of the encapsulated peptide to HepG2 and HSKM cells was confirmed by its prolonged effect on stimulation of glucose uptake (>6 h). The peptide improved glucose clearance in both wild-type and Ob/Ob mice; it lowered blood glucose levels and suppressed glucose-stimulated insulin secretion. IRK activity was stimulated in the liver of treated mice and in cultured cells. The peptide potentiated function of IRK’s downstream effector, Akt-GSK3-(α,β)axis. Thus, P2-based approach can be used for improving glucose uptake by cells. Also, it allows for screening peptidesin vitroand in animal models for treatment of diabetes.

scholarly journals Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways

2018 ◽  
Vol 29 (6) ◽  
pp. 1720-1730 ◽  
Author(s):  
Miriam Zacchia ◽  
Xuefei Tian ◽  
Enrica Zona ◽  
Robert J. Alpern ◽  
Patricia A. Preisig
Keyword(s):  
Proximal Tubule ◽  
Signaling Pathways ◽  
Cultured Cells ◽  
Wild Type ◽  
Experimental Conditions ◽  
Opossum Kidney ◽  
Citrate Transporter ◽  
Stimulation Of

Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2−/−) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro. Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2−/− mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro. Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

THE EFFECT OF GROWTH HORMONE AND OF CORTISOL ON THE RESPONSE OF ISOLATED RAT DIAPHRAGM TO THE STIMULATING EFFECT OF INSULIN ON GLUCOSE UPTAKE AND ON INCORPORATION OF AMINO ACIDS INTO PROTEIN

1959 ◽  
Vol 18 (4) ◽  
pp. 395-408 ◽  
Author(s):  
K. L. MANCHESTER ◽  
P. J. RANDLE ◽  
F. G. YOUNG
Keyword(s):  
Amino Acids ◽  
Growth Hormone ◽  
Protein Synthesis ◽  
Glucose Uptake ◽  
Carbohydrate Metabolism ◽  
Rat Diaphragm ◽  
Pituitary Growth Hormone ◽  
Isolated Rat ◽  
Stimulation Of

SUMMARY 1. The effect of hypophysectomy, or of adrenalectomy, and injection of pituitary growth hormone (GH) or of cortisol, on the uptake of glucose and the incorporation of glycine into protein by isolated rat diaphragm, and the effect of the addition of insulin in vitro on these processes, has been studied. 2. Both hypophysectomy and adrenalectomy raised the uptake of glucose by isolated diaphragm, while treatment of the intact or of the hypophysectomized rat with GH, or of the intact or of the adrenalectomized rat with cortisol, depressed it. Although hypophysectomy and adrenalectomy did not influence the additional glucose uptake induced by 200 mu./ml. of insulin in vitro, both these operations enhanced the effect of 0·1–1·0 mu./ml. of insulin on glucose uptake by diaphragm in vitro. Treatment of the rat with GH or cortisol diminished the rise in glucose uptake of diaphragm induced by 0·1–1·0 mu./ml. insulin. 3. Hypophysectomy depressed, and administration of GH to the intact or hypophysectomized rat raised, the incorporation of glycine into protein of the isolated diaphragm, but neither of these operations altered the magnitude of the stimulation of incorporation induced by 1·0 mu./ml. insulin. 4. Adrenalectomy raised, and administration of cortisol to the intact or adrenalectomized rat depressed, the incorporation of glycine into protein of the isolated diaphragm; adrenalectomy enhanced, the injection of cortisol diminished, the effect of 1·0 mu./ml. insulin on these processes. 5. The possibility that GH directs insulin towards the stimulation of protein synthesis, in part by restraining the action of insulin on carbohydrate metabolism, is discussed.

Kinase activity of mutant LRRK2 manifests differently in hetero-dimeric vs. homo-dimeric complexes

2019 ◽  
Vol 476 (3) ◽  
pp. 559-579 ◽  
Author(s):  
Emmanouela Leandrou ◽  
Eliana Markidi ◽  
Anna Memou ◽  
Katerina Melachroinou ◽  
Elisa Greggio ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Cultured Cells ◽  
Kinase Domain ◽  
Small Gtpase ◽  
Wild Type ◽  
Risk Variant ◽  
Dimeric Complexes ◽  
Mutant Lrrk2 ◽  
Mutant Forms

Abstract The Parkinson's disease (PD) protein leucine-rich repeat kinase 2 (LRRK2) exists as a mixture of monomeric and dimeric species, with its kinase activity highly concentrated in the dimeric conformation of the enzyme. We have adapted the proximity biotinylation approach to study the formation and activity of LRRK2 dimers isolated from cultured cells. We find that the R1441C and I2020T mutations both enhance the rate of dimer formation, whereas, the G2019S kinase domain mutant is similar to WT, and the G2385R risk factor variant de-stabilizes dimers. Interestingly, we find a marked departure in the kinase activity between G2019S–LRRK2 homo-dimers and wild-type-G2019S hetero-dimers. While the homo-dimeric G2019S–LRRK2 exhibits the typical robust enhancement of kinase activity, hetero-dimers comprised of wild-type (WT) and G2019S–LRRK2 exhibit kinase activity similar to WT. Dimeric complexes of specific mutant forms of LRRK2 show reduced stability following an in vitro kinase reaction, in LRRK2 mutants for which the kinase activity is similar to WT. Phosphorylation of the small GTPase Rab10 follows a similar pattern in which hetero-dimers of WT and mutant LRRK2 show similar levels of phosphorylation of Rab10 to WT homo-dimers; while the levels of pRab10 are significantly increased in cells expressing mutant homo-dimers. Interestingly, while the risk variant G2385R leads to a de-stabilization of LRRK2 dimers, those dimers possess significantly elevated kinase activity. The vast majority of familial LRRK2-dependent PD cases are heterozygous; thus, these findings raise the possibility that a crucial factor in disease pathogenesis may be the accumulation of homo-dimeric mutant LRRK2.

IN VITRO METHODS FOR THE STUDY OF THE ADENOHYPOPHYSIAL FUNCTIONS TO SECRETE GONADOTROPHIN

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S27-S40 ◽  
Author(s):  
T. Kobayashi ◽  
T. Kigawa ◽  
M. Mizuno ◽  
T. Watanabe
Keyword(s):  
Cell Culture ◽  
Organ Culture ◽  
Cultured Cells ◽  
Cell Sheet ◽  
Short Term ◽  
Hypothalamic Extract ◽  
Numerous Cell ◽  
Single Cell Culture ◽  
Almost All

ABSTRACT There are several in vitro methods to analyse the function of the adenohypophysis or the mechanisms of its regulation. The present paper deals with single cell culture, organ culture and short term incubation techniques by which the morphology and gonadotrophin-secreting function of the adenohypophysis were studied. In trypsin-dispersed cell culture, the adenohypophysial cells showed extensive propagation to form numerous cell colonies and finally develop into a confluent monolayer cell sheet covering completely the surface of culture vessels. Almost all of the cultured cells, however, became chromophobic, at least at the end of the first week of cultivation, when gonadotrophin was detectable neither in the culture medium nor in the cells themselves. After the addition of the hypothalamic extract, gonadotrophin became detectable again, and basophilic or PAS-positive granules also reappeared within the cells, suggesting that the gonadotrophs were stimulated by the extract to produce gonadotrophin. In organ culture and short term incubation, the incorporation of [3H] leucine into the adenohypophysial cells in relation to the addition of hypothalamic extract was examined. It was obvious that the ability to incorporate [3H] leucine into the gonadotrophs in vitro was highly dependent upon the presence of the hypothalamic extract.

The URNA Genes of Herpesvirus Saimiri (Strain C488) Are Dispensable for Transformation of Human T Cells In Vitro

1999 ◽  
Vol 73 (12) ◽  
pp. 10551-10555 ◽  
Author(s):  
Armin Ensser ◽  
André Pfinder ◽  
Ingrid Müller-Fleckenstein ◽  
Bernhard Fleckenstein
Keyword(s):  
Cell Culture ◽  
Herpesvirus Saimiri ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Human T Cell ◽  
Human T Cells ◽  
Small Nuclear Rnas ◽  
T Cell Lines

ABSTRACT The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.

A New In Vitro Model for Predicting the Toxicity of Cosmetic Products

1992 ◽  
Vol 20 (1) ◽  
pp. 138-143
Author(s):  
Maria Carrara ◽  
Lorenzo Cima ◽  
Roberto Cerini ◽  
Maurizio Dalle Carbonare
Keyword(s):  
Cell Culture ◽  
Cultured Cells ◽  
Neutral Red ◽  
Total Protein Content ◽  
Cosmetic Products ◽  
Cell Culture Insert ◽  
A Cell ◽  
Vitro Model ◽  
Cosmetic Emulsions

A method has been developed whereby cosmetic products which are not soluble in water or in alcohol can be brought into contact with cell cultures by being placed in a cell culture insert, which is then placed in the cell culture well. Preliminary experiments were carried out with L929 cells, and cytotoxicity was evaluated by measuring neutral red uptake and the total protein content of treated cultured cells. Encouraging results were obtained in comparisons of three cosmetic emulsions and of one emulsion containing a range of concentrations of two preservatives, Kathon CG and Bronopol.

scholarly journals Mechanical Strain-Enabled Reconstitution of Dynamic Environment in Organ-on-a-Chip Platforms: A Review

Micromachines ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 765
Author(s):  
Qianbin Zhao ◽  
Tim Cole ◽  
Yuxin Zhang ◽  
Shi-Yang Tang
Keyword(s):  
Cell Culture ◽  
Shear Stress ◽  
Cultured Cells ◽  
Mechanical Strain ◽  
3D Cell Culture ◽  
Small Scale ◽  
Mechanical Stimuli ◽  
Human Organ ◽  
Organ On A Chip

Organ-on-a-chip (OOC) uses the microfluidic 3D cell culture principle to reproduce organ- or tissue-level functionality at a small scale instead of replicating the entire human organ. This provides an alternative to animal models for drug development and environmental toxicology screening. In addition to the biomimetic 3D microarchitecture and cell–cell interactions, it has been demonstrated that mechanical stimuli such as shear stress and mechanical strain significantly influence cell behavior and their response to pharmaceuticals. Microfluidics is capable of precisely manipulating the fluid of a microenvironment within a 3D cell culture platform. As a result, many OOC prototypes leverage microfluidic technology to reproduce the mechanically dynamic microenvironment on-chip and achieve enhanced in vitro functional organ models. Unlike shear stress that can be readily generated and precisely controlled using commercial pumping systems, dynamic systems for generating proper levels of mechanical strains are more complicated, and often require miniaturization and specialized designs. As such, this review proposes to summarize innovative microfluidic OOC platforms utilizing mechanical actuators that induce deflection of cultured cells/tissues for replicating the dynamic microenvironment of human organs.

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3336-3344 ◽  
Author(s):  
Anu Laitala ◽  
Ellinoora Aro ◽  
Gail Walkinshaw ◽  
Joni M. Mäki ◽  
Maarit Rossi ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mrna Level ◽  
Hypoxia Inducible Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Α Subunit ◽  
Hepcidin Expression ◽  
In The Wild ◽  
Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

scholarly journals Propagation of bovine spermatogonial stem cells in vitro

Reproduction ◽  
2008 ◽  
Vol 136 (5) ◽  
pp. 543-557 ◽  
Author(s):  
Pedro M Aponte ◽  
Takeshi Soda ◽  
Katja J Teerds ◽  
S Canan Mizrak ◽  
Henk J G van de Kant ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Cultured Cells ◽  
Somatic Cells ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Type A Spermatogonia ◽  
Stimulation Of

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study thein vitrobehavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

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