Role of kallikrein-kininogen system in insulin-stimulated glucose transport after muscle contractions

2002 ◽  
Vol 92 (2) ◽  
pp. 657-664 ◽  
Author(s):  
C. L. Dumke ◽  
J. Kim ◽  
E. B. Arias ◽  
G. D. Cartee
Keyword(s):  
Glucose Transport ◽  
Serum Proteins ◽  
Muscle Contractions ◽  
Rat Serum ◽  
Serum Free ◽  
Bradykinin Receptor ◽  
Contraction Effect ◽  
Kallikrein Inhibitors

Serum proteins [molecular weight (MW) > 10,000] are essential for increased insulin-stimulated glucose transport after in vitro muscle contractions. We investigated the role of the kallikrein-kininogen system, including bradykinin, which is derived from kallikrein (MW > 10,000)-catalyzed degradation of serum protein kininogen (MW > 10,000), on this contraction effect. In vitro electrical stimulation of rat epitrochlearis muscles was performed in 1) rat serum ± kallikrein inhibitors; 2) human plasma (normal or kallikrein-deficient); 3) rat serum ± bradykinin receptor-2 inhibitors; or 4) serum-free buffer ± bradykinin. 3- O-methylglucose transport (3-MGT) was measured 3.5 h later. Serum ± kallikrein inhibitors tended ( P = 0.08) to diminish postcontraction insulin-stimulated 3-MGT. Contractions in normal plasma enhanced insulin-stimulated 3-MGT vs. controls, but contractions in kallikrein-deficient plasma did not. Supplementing rat serum with bradykinin receptor antagonist HOE-140 during contraction did not alter insulin-stimulated 3-MGT. Muscles stimulated to contract in serum-free buffer plus bradykinin did not have enhanced insulin-stimulated 3-MGT. Bradykinin was insufficient for postcontraction-enhanced insulin sensitivity. However, results with kallikrein inhibitors and kallikrein-deficient plasma suggest kallikrein plays a role in this improved insulin action.

The role of the visceral yolk sac in mediating protein utilization by rat embryos cultured in vitro

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 223-234
Author(s):  
Stuart J. Freeman ◽  
Felix Beck ◽  
John B. Lloyd
Keyword(s):  
Serum Proteins ◽  
Yolk Sac ◽  
Rat Serum ◽  
Pregnant Rats ◽  
Digestion Product ◽  
Background Levels ◽  
Visceral Yolk Sac ◽  
Chorioallantoic Placenta

Conceptuses from 9·5-day pregnant rats have been cultured for 48 h in heat-inactivated homologous serum. Embryonic development was normal. The protein contents of embryos and visceral yolk sacs after different periods of culture were recorded. When 125-labelled polyvinylpyrrolidone or [3H]dextran were added to the culture serum, radioactivity was accumulated by the yolk sac, but only background levels were detected in the embryo itself. The amount of radioactivity found in the yolk sac varied with the length of the interval before harvesting during which 125 I-labelled PVP or [3H]dextran was present. When formaldehyde-denatured 125 I-labelled bovine serum albumin was added to the culture serum, little radioactivity accumulated in the yolk sac and only background levels were found in the embryo. Trichloroacetic acid-soluble radioactivity steadily appeared in the culture serum, however. When conceptuses were cultured in glucose- and vitamin-supplemented dialysed serum from rats injected 2 h previously with [3H]leucine, radioactivity was found in both embryos and yolk sacs. The amount of radioactivity in these tissues increased with duration of exposure to 3H-labelled serum proteins. After short exposures little of the yolk sac and embryonic radioactivity was acid-insoluble, but this proportion increased with duration of exposure. These results are interpreted as follows. Intact macromolecules cannot enter the cells of the embryo itself, but are captured by pinocytosis into the cells of the visceral yolk-sac endoderm. Indigestible macromolecules such as 125 I-labelled polyvinylpyrrolidone and [3H]- dextran accumulate within the yolk-sac lysosomes, but proteins are digested there by the lysosomal enzymes. The radiolabelled digestion product of 125 I-labelled bovine albumin is [125 I]iodotyrosine, which cells cannot utilize and so is excreted into the culture serum. The labelled digestion product of the 3H-labelled rat serum proteins is [3H]leucine, which is used for protein synthesis in both embryo and yolk sac. The experiments provide direct evidence for the long-suspected role of the yolk sac in mediating embryonic nutrition in the period of development prior to the establishment of a functional chorioallantoic placenta.

INCREASE OF SERUM FREE THYROXINE FOLLOWING THE ADMINISTRATION OF THIOCYANATE AND OTHER ANIONS IN VIVO AND IN VITRO

1974 ◽  
Vol 75 (4) ◽  
pp. 707-716 ◽  
Author(s):  
N. Michajlovskij ◽  
P. Langer
Keyword(s):  
Time Course ◽  
Linear Increase ◽  
Free Thyroxine ◽  
Peroral Administration ◽  
Rat Serum ◽  
Serum Free ◽  
Serum Free Thyroxine

ABSTRACT Following the addition of thiocyanate (SCN−) to human and rat serum of 80 mg SCN−/ 100 ml (initial concentration before dialysis) a marked increase in serum free thyroxine (FT4) was found. The addition of equivalent doses of other antihyroid anions or permanganate showed a similar increase of FT4 in the order: BF4− < CIO4− = SCN− < MnO4−. The increase in the rat serum was greater than that in human serum. After peroral administration of these anions to the rats in amounts equal to 30 mg SCN−/rat the increase of FT4 was still higher. The addition of increased doses of SCN− to the human and rat sera (5-80 and up to 1600 mg SCN−/100 ml) caused a linear increase of FT4. Similarly, after the administration of various doses of SCN− to rats (5-30 mg SCN−/rat) a linear dose-response of the FT4 level in serum was observed. The time-course of the increase of % FT4 and absolute FT4 (AFT4) level after a single administration of SCN− to rats coincided with the increase of the serum SCN− level. However, after the disappearance of SCN− from the serum the % FT4 returned to the initial value, while the AFT4 was decreased. The effect of these anions probably consists in the competitive displacement of thyroxine from serum thyroxine-binding proteins. These and previous results suggested that thiocynate influences the plasma protein-thyroxine equilibrium and the possible role of an increased free thyroxine level on the action of thyroxine on various tissues and the hypothalamo-pituitary-thyroid feed-back is discussed.

Reversal of the exercise-induced increase in muscle permeability to glucose

1987 ◽  
Vol 253 (4) ◽  
pp. E331-E335 ◽  
Author(s):  
D. A. Young ◽  
H. Wallberg-Henriksson ◽  
M. D. Sleeper ◽  
J. O. Holloszy
Keyword(s):  
Protein Synthesis ◽  
Glucose Transport ◽  
Glycogen Synthesis ◽  
Carbohydrate Restriction ◽  
Rat Serum ◽  
Low Concentration ◽  
Carbohydrate Feeding ◽  
Exercise Induced

Exercise is associated with an increase in permeability of muscle to glucose that reverses slowly (h) in fasting rats during recovery. Previous studies showed that carbohydrate feeding speeds and carbohydrate restriction slows reversal of the exercise-induced increase in glucose uptake. This study was designed to evaluate the roles of glucose transport, glycogen synthesis, and protein synthesis in the reversal process in rat epitrochlearis muscle. In contrast to recovery in vivo, when muscles were incubated without insulin in vitro, the exercise-induced increase in muscle permeability to sugar reversed rapidly regardless of whether glucose transport or glycogen synthesis occurred. Inhibition of protein synthesis did not prevent the reversal. Addition of 33% rat serum or a low concentration of insulin to the incubation medium markedly slowed reversal in vitro. We conclude that 1) prolonged persistence of the increased permeability of mammalian muscle to glucose after exercise requires a low concentration of insulin, and 2) reversal of the increase in permeability does not require glucose transport, glycogen synthesis, or protein synthesis.

scholarly journals Enhancement of In Vitro Growth of Pathogenic Bacteria by Norepinephrine: Importance of Inoculum Density and Role of Transferrin

2006 ◽  
Vol 72 (7) ◽  
pp. 5097-5099 ◽  
Author(s):  
Phyllis M. O'Donnell ◽  
Hernan Aviles ◽  
Mark Lyte ◽  
Gerald Sonnenfeld
Keyword(s):  
Bacterial Growth ◽  
Pathogenic Bacteria ◽  
Inoculum Density ◽  
In Vitro Growth ◽  
Stress Hormone ◽  
Serum Free ◽  
Free Media

ABSTRACT Norepinephrine is a stress hormone that enhances bacterial growth. We examined the effects of a small inoculum on the norepinephrine-induced growth of species previously reported to be unaffected by norepinephrine. The results indicated that a reduced inoculum density is essential for observing norepinephrine-induced effects. Additional studies using serum-free media suggested that transferrin plays a role in norepinephrine-induced growth.

268 ROLE OF ATPase MOTOR CYTOPLASMIC DYNEIN AND PRIMARY CILIA IN TESTICULAR MORPHOGENESIS

2015 ◽  
Vol 27 (1) ◽  
pp. 223
Author(s):  
C. Dores ◽  
I. Dobrinski
Keyword(s):  
Primary Cilia ◽  
Somatic Cells ◽  
Treated Group ◽  
Cytoplasmic Dynein ◽  
Control Group ◽  
Tubule Formation ◽  
Serum Free ◽  
Free Media

In vertebrates, the primary cilium is a nearly ubiquitous organelle present in somatic cells, but little is known about its function in the male gonad. We investigated the role of primary cilia in testis cells using in vitro formation of seminiferous tubules and in vitro culture of testicular somatic cells by inhibiting the primary cilium with CiliobrevinD, a cell-permeable, reversible chemical modulator that inhibits the major component of the organelle: ATPase motor cytoplasmic dynein. We analysed in vitro cultures for the presence of primary cilia and the activation of hedgehog signalling through translocation of Gli2 to the nuclei; in vitro tubule formation was evaluated by length and width of tubules formed. Methods: testicular cells were harvested from neonatal pigs by 2-step enzymatic digestion. Cells (50 × 106 mL–1) were plated on 100 mm Petri dishes in 15 mL of DMEM + 5% FBS + 50 U of penicillin and incubated at 37°C in 5% CO2 in air overnight, cells remaining in suspension and those slightly attached were removed and the somatic cells attached were trypsinized to obtain a single cell suspension, and then submitted to two different protocols: in vitro culture (A) or in vitro tubule formation (B), n = 5 replicates each. For A, somatic cells were replated on coverslips in 24-well plates and cultured in serum free media for 48 h, then for the treated group, 10 mM of CiliobrevinD was added for 24 h, attached cells from control and treated groups were fixed in 4% PFA and characterised by immunocytochemistry for ARL13B, Vimentin, and Gli2. For B: 1 × 106 cells were added to 24-well plates coated with 1 : 1 diluted Matrigel, the control group was kept in serum free media and to the treated group was added 20 mM CiliobrevinD at Day 0. Results: A) primary cilia were present in 89.3 ± 2.3% of cells cultured in serum-free media for the control group and Gli2 was located in the nuclei of 90.2 ± 1.2% of cells; in the CiliobrevinD-treated group the percentage of primary cilia decreased (P < 0.05) to 3.1 ± 2.5% and nuclear Gli2 to 3.9 ± 0.7; B) tubules formed in the control group were significantly longer and wider than the ones formed when CiliobrevinD was added (9.91 ± 0.35 v. 5.540 ± 1.08 mm and 339.8 ± 55.78 v. 127.2 ± 11.9 µm, respectively, P < 0.05 by Student's t-test). In conclusion, the inhibition of ATPase motor cytoplasmic dynein perturbs formation of primary cilia in testicular somatic cells, blocks Hedgehog signalling, and impairs in vitro tubule formation. Therefore, primary cilia on testicular somatic cells appear to be essential for testicular morphogenesis.Research was supported by 5 R01 OD016575-13.

Role of Organic Phosphate in Mineralization of Bone in vitro

1981 ◽  
Vol 60 (C) ◽  
pp. 1586-1586 ◽  
Author(s):  
H. C. Tenenbaum
Keyword(s):  
Defined Medium ◽  
Organic Phosphate ◽  
Limiting Factor ◽  
Serum Free ◽  
Osteogenic Cells ◽  
Inorganic Phosphates

Calvarial periostea were dissected from 17-day-old embryonic chicks and folded with the osteogenic cells in apposition. The folded explains were cultured for up to six d on serum and plasma clots or in serum-free hormone-supplemented completely-defined medium. Osteoid consistently formed in such cultures in both types of media, and this osteoid mineralized when appropriate levels of β-glycerophosphate were added to each type of medium. The data presented suggest that the levels of organic phosphate might be more important than inorganic phosphates as a limiting factor in the initiation of mineralization of bone in vitro.

Serum interspecies differences in metabolic pathways of bradykinin and [des-Arg9]BK: influence of enalaprilat

1996 ◽  
Vol 271 (4) ◽  
pp. H1340-H1347 ◽  
Author(s):  
A. Decarie ◽  
P. Raymond ◽  
N. Gervais ◽  
R. Couture ◽  
A. Adam
Keyword(s):  
Metabolic Pathways ◽  
Neutral Endopeptidase ◽  
Thrombin Inhibitor ◽  
Rat Serum ◽  
Highly Sensitive ◽  
Significant Difference ◽  
Hydrolysis Of

Among the different enzymes responsible for the metabolism of bradykinin (BK), three peptidases look relevant in vivo: kininase I (KI), which transforms BK into its active metabolite, [des-Arg9]BK; kininase II (KII); and neutral endopeptidase, which inactivate BK and [des-Arg9]BK. The in vitro incubation of BK and [des-Arg9]BK in the serum of four species with or without enalaprilat and the quantification of the immunoreactivity of both peptides at different time intervals allowed the measurement of the kinetic parameters characterizing their metabolic pathways. Highly sensitive chemiluminescent enzyme immunoassays were used to measure the residual concentrations of BK and [des-Arg9]BK. Half-life (t1/2) of BK showed significant difference among species: rats (10 +/- 1 s) = dogs (13 +/- 1 s) < rabbits (31 +/- 1 s) < humans (49 +/- 2 s). t1/2 values of [des-Arg9]BK were also species dependent: rats (96 +/- 6 s) < < rabbits (314 +/- 6 s) = dogs (323 +/- 11 s) = humans (325 +/- 12 s). Enalaprilat significantly prevented the rapid BK and [des-Arg9]BK degradation in all species except that of [des-Arg9]BK in rat serum. Relative amount of BK hydrolyzed by serum KII was given as follows: rabbits (93.7 +/- 14.8%) = rats (83.6 +/- 6.7%) = humans (76.0 +/- 7.5%) > dogs (50.0 +/- 3.9%). Its importance in the hydrolysis of [des-Arg9]BK was 5.2 +/- 0.5% in rats < < 33.9 +/- 1.5% in humans < 52.0 +/- 1.1% in rabbits < 65.1 +/- 3.4% in dogs. The participation of serum KI in the transformation of BK into [des-Arg9]BK was dogs (67.2 +/- 5.3%) > > humans (3.4 +/- 1.2%) = rabbits (1.8 +/- 0.2%) = rats (1.4 +/- 0.3%). Finally, no significant difference on t1/2 values for BK and [des-Arg9]BK could be demonstrated between serum and plasma treated with either sodium citrate or a thrombin inhibitor. These results revealed striking species differences in the serum metabolism of kinins that could address at least partially some of the controversial data related to the cardioprotective role of kinins.

scholarly journals Sorting of GLUT4 into its insulin-sensitive store requires the Sec1/Munc18 protein mVps45

2013 ◽  
Vol 24 (15) ◽  
pp. 2389-2397 ◽  
Author(s):  
Jennifer Roccisana ◽  
Jessica B. A. Sadler ◽  
Nia J. Bryant ◽  
Gwyn W. Gould
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Vitro Assay ◽  
Protein Receptor ◽  
Storage Vesicles ◽  
Storage Vesicle ◽  
Impaired Insulin ◽  
Endosomal Pathway

Insulin stimulates glucose transport in fat and muscle cells by regulating delivery of the facilitative glucose transporter, glucose transporter isoform 4 (GLUT4), to the plasma membrane. In the absence of insulin, GLUT4 is sequestered away from the general recycling endosomal pathway into specialized vesicles, referred to as GLUT4-storage vesicles. Understanding the sorting of GLUT4 into this store is a major challenge. Here we examine the role of the Sec1/Munc18 protein mVps45 in GLUT4 trafficking. We show that mVps45 is up-regulated upon differentiation of 3T3-L1 fibroblasts into adipocytes and is expressed at stoichiometric levels with its cognate target–soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 16. Depletion of mVps45 in 3T3-L1 adipocytes results in decreased GLUT4 levels and impaired insulin-stimulated glucose transport. Using sub­cellular fractionation and an in vitro assay for GLUT4-storage vesicle formation, we show that mVps45 is required to correctly traffic GLUT4 into this compartment. Collectively our data reveal a crucial role for mVps45 in the delivery of GLUT4 into its specialized, insulin-regulated compartment.

scholarly journals THE SECRETORY PATHWAYS OF RAT SERUM GLYCOPROTEINS AND ALBUMIN

1972 ◽  
Vol 52 (2) ◽  
pp. 231-245 ◽  
Author(s):  
Colvin M. Redman ◽  
M. George Cherian
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Rough Endoplasmic Reticulum ◽  
Serum Proteins ◽  
Rat Serum ◽  
Secretory Pathways ◽  
Specific Antisera ◽  
Microsome Fraction

These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein. N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membrane-attached polysomes and not into proteins from free polysomes. Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes. Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae. Albumin was mostly recovered (98%) in the cisternae, with negligible amounts in the membranes. To determine whether the radio-active sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the 14C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins. The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae.

scholarly journals Role of facilitative glucose uptake in the glucose-inorganic phosphate-mediated retardation and inhibition of development in different strains of mouse embryos

Reproduction ◽  
2002 ◽  
pp. 691-700 ◽  
Author(s):  
L Scott ◽  
DG Whittingham
Keyword(s):  
Glucose Uptake ◽  
Glucose Transport ◽  
Inorganic Phosphate ◽  
Mouse Embryos ◽  
Concentration Gradients ◽  
Morula Stage ◽  
Different Strains ◽  
Hybrid Development

Mouse embryos from different strains develop differently in vitro depending on the composition of the culture medium, and in particular on the presence or absence of glucose and inorganic phosphate. Glucose is both stimulatory and inhibitory in certain conditions. Glucose uptake by cells can be passive, down concentration gradients, or active, through sodium driven pumps, or can occur through facilitative transport. This study investigated the effects of inhibition of facilitative glucose transport on the glucose-inorganic phosphate-mediated blocks in development in three different strains of mouse embryo, CF-1, CD-1 and an F2 hybrid. Development of CF-1 and CD-1 embryos is blocked in medium containing glucose and inorganic phosphate but not in medium containing glucose alone, and F2 embryos are not affected. Inhibition of facilitated glucose transport to the eight-cell-morula stage in CF-1 and CD-1 embryos resulted in development in medium containing both glucose and inorganic phosphate, indicating that the prevention of facilitative glucose uptake can overcome the developmental block. Removal of inhibition before the eight-cell-morula stage resulted in total arrest of CF-1 embryos and minimum development of CD-1 embryos. F2 embryos are not affected by inorganic phosphate and glucose and showed no response to the transporter inhibitor at any stage. These data support the contention that facilitated glucose transport is active in embryos, is phosphate-dependent and that its inhibition can overcome the glucose-inorganic phosphate-mediated developmental blocks in mouse embryos.

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