Endogenous effectors of human liver glycogen phosphorylase modulate effects of indole-site inhibitors

2005 ◽  
Vol 289 (3) ◽  
pp. E366-E372 ◽  
Author(s):  
Nacide Ercan-Fang ◽  
Miriam R. Taylor ◽  
Judith L. Treadway ◽  
Carolyn B. Levy ◽  
Paul E. Genereux ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Human Liver ◽  
Glycogen Phosphorylase ◽  
Liver Glycogen ◽  
Inhibitory Effect ◽  
Small Molecular Weight ◽  
Glucose Lowering ◽  
Glycogen Phosphorylase Inhibitors

Phosphorylase is regulated by a number of small-molecular-weight effectors that bind to three sites on the enzyme. Recently, a fourth site referred to as the indole-inhibitor site has been identified. Synthetic compounds bind to the site and inhibit activity. However, the effects of these compounds in the presence of other endogenous effectors are unknown. We have determined the effects of four indole derivative glycogen phosphorylase inhibitors (GPI) on recombinant human liver glycogen phosphorylase a activity. The GPIs tested were all potent inhibitors. However, the endogenous inhibitors (glucose, ADP, ATP, fructose 1-phosphate, glucose 6-phosphate, UDP-glucose) and the activator (AMP) markedly reduced the inhibitory effect of GPIs. Consistent with these in vitro findings, the IC50 for the inhibition of glycogenolysis in cells and the liver drug concentration associated with glucose-lowering activity in diabetic ob/ ob mice in vivo were also significantly higher than those determined in in vitro enzyme assays. The inhibitory effect of indole-site effectors is modulated by endogenous small-molecular-weight effectors of phosphorylase a activity. However, at higher concentrations (10–30 μM), the GPI effect was dominant and resulted in inhibition of phosphorylase a activity irrespective of the presence or absence of the other modulators of the enzyme.

scholarly journals In vitro inhibitory effect of obtusofolin on the activity of CYP3A4, 2C9, and 2E1

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Na Liu ◽  
Ping Chen ◽  
Xiaojun Du ◽  
Junxia Sun ◽  
Shasha Han
Keyword(s):  
Human Liver ◽  
Competitive Inhibition ◽  
Liver Microsomes ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Cytochrome P450 Enzymes ◽  
Inhibition Model ◽  
Dose Dependent

Abstract Background Obtusofolin is the major active ingredient of Catsia tora L., which possesses the activity of improving eyesight and protecting the optic nerve. Investigation on the interaction of obtusofolin with cytochrome P450 enzymes (CYP450s) could provide a reference for the clinical application of obtusofolin. Methods The effect of obtusofolin on the activity of CYP450s was investigated in the presence of 100 μM obtusofolin in pooled human liver microsomes (HLMs) and fitted with the Lineweaver–Burk plots to characterize the specific inhibition model and kinetic parameters. Results Obtusofolin was found to significantly inhibited the activity of CYP3A4, 2C9, and 2E1. In the presence of 0, 2.5, 5, 10, 25, 50, and 100 μM obtusofolin, the inhibition of these CYP450s showed a dose-dependent manner with the IC50 values of 17.1 ± 0.25, 10.8 ± 0.13, and 15.5 ± 0.16 μM, respectively. The inhibition of CYP3A4 was best fitted with the non-competitive inhibition model with the Ki value of 8.82 μM. While the inhibition of CYP2C9 and 2E1 was competitive with the Ki values of 5.54 and 7.79 μM, respectively. After incubating for 0, 5, 10, 15, and 30 min, the inhibition of CYP3A4 was revealed to be time-dependent with the KI value of 4.87 μM− 1 and the Kinact value of 0.0515 min− 1. Conclusions The in vitro inhibitory effect of obtusofolin implying the potential drug-drug interaction between obtusofolin and corresponding substrates, which needs further in vivo validations.

scholarly journals Human liver glycogen phosphorylase inhibitors bind at a new allosteric site

2000 ◽  
Vol 7 (9) ◽  
pp. 677-682 ◽  
Author(s):  
Virginia L Rath ◽  
Mark Ammirati ◽  
Dennis E Danley ◽  
Jennifer L Ekstrom ◽  
E Michael Gibbs ◽  
...  
Keyword(s):  
Human Liver ◽  
Glycogen Phosphorylase ◽  
Liver Glycogen ◽  
Allosteric Site ◽  
Glycogen Phosphorylase Inhibitors

Complexing of Low Mr-Weight Human Urokinase In Rat Serum And Its Degradation Into Fragments Of Very Small Molecular Weight In Vivo But Not In Vitro

1981 ◽  
Author(s):  
Ph Schneider ◽  
M Ruegg ◽  
F Bachmann
Keyword(s):  
Molecular Weight ◽  
Inferior Vena ◽  
Vena Cava ◽  
Albino Rats ◽  
Small Molecular Weight ◽  
Rat Serum ◽  
Blood Samples ◽  
Sds Page

Highly purified lew molecular weight urokinase (LMR-UK), moving on SDS-PAGE (reduced and nan-reduced) as a single band of 32 kdalton, was labelled with 125I by the chlora- mine-T method. 106 cpn of this 125I-LMr-UK (94% TCA preci- pitable) were injected into the inferior vena cava of la- par atomized albino rats, which were maintained at 37°C. Blood samples were collected by cardiac puncture 5, 30 and 90 min respectively after the injection. Serun, obtained from these samples, was fractionated on a Sephadex G-100 column, calibrated with proteins of known Mr. Radioactivity was measured in the collected fractions.In the 5 min sample, the radioactivity was distributed in 2 peaks, corresponding to 32 kdalton and to < 70 kdalton respectively. In the 30 min sample, the distribution was characterized by a diminution of the 32 kdalton peak and the appearance of a third peak corresponding to a Mr of < 4 kdalton. In the 90 min sample, the LMr-UK peak had disappeared almost completely. About 40% of the 125I-activity was present in a skewed high Mr peak with a broad maximum in the 85-100 kdalton region; ≥ 60% of the 125I-activity was recovered in late fractions corresponding to < 4 kdalton. In control experiments, pooled rat serum was incubated in vitro with 125I-LMr-UK for 5, 30 and 90 min respectively and samples were fractionated on the same column. The radioactivity distribution shewed only the 32 and > 70 kdalton peaks, but no < 4 kdalton peak.These results suggest that LMr-UK is complexed to a carrier protein, both in vivo and in vitro, but that it is degraded into small fragments in vivo only. Attempts to characterize the nature of these complexes are in progress.

On-Line Hemodiafiltration with Pre- and Postdilution: A Comparison of Efficacy

1997 ◽  
Vol 20 (2) ◽  
pp. 81-90 ◽  
Author(s):  
P. Ahrenholz ◽  
R.E. Winkler ◽  
W. Ramlow ◽  
M. Tiess ◽  
W. Müller
Keyword(s):  
Molecular Weight ◽  
Theoretical Description ◽  
Convective Transport ◽  
Small Molecular Weight ◽  
Dialysate Flow ◽  
Significant Difference ◽  
On Line ◽  
The Impact

Since the introduction of on-line substituate preparation, high substituate rates (Qs) in pre- and postdilution for hemodiafiltration (HDF) procedures can be realized. During postdilution HDF (POD-HDF) and additional convective removal is possible, but in vivo Qs is limited to approx. 1/3Qb (bloodflow). With predilution HDF (PRD-HDF) higher Qs and therefore high convective transport rates by ultrafiltration can be reached. On the other hand the blood concentration is diminished by predilution. Further decrease of the diffusive transport is caused by reduced dialysate flow Qd due to separation of the substituate from the dialysate (Fresenius 4008 On-Line HDF, Gambro AK100 Ultra). The theoretical description of the combined diffusive-convective transport is limited to 1-dimensional models and small UF-rates. Therefore for practical and theoretical purposes the assessment of the efficacy of on-line PRD-HDF and POD-HDF in different molecular weight ranges is desirable. By means of in vitro experiments the effective clearances Keff of hemodialysis (HD, dialyzer: Fresenius F60) for urea, creatinine, vitamin B12 and inulin were compared with measured and theoretical Keff of POD- and PRD-HDF. The theoretical expectation is confirmed that Keff for small molecular weight substances decreases slightly with PRD-HDF and increases for larger molecules. In the case of POD-HDF Keff for small molecular weight substances increases slightly and strongly for larger molecules. In vivo experiments were performed to measure the real substance removal from patient's blood and to figure out the impact of dialysate flow (collection of the used dialysate during the 1. treatment hour and concentration measurements for urea, creatinine, phosphate, ß2-MG). The results show that the substraction of Qs from Qd reduces Keff for urea, creatinine and phosphate but not for ß2-MG. PRD-HDF with Qd = 500 ml/min is significantly less effective for small molecules than HD. There is no significant difference of Keff for urea, creatinine, phosphate during HD and PRD-HDF with Qd = 800 ml/min, but a significant increase of 10-15% for POD-HDF Keff for ß2-MG increases by 75% for PRD-HDF and 95% for POD-HDF compared with HD (Qd = 500 ml/min).

Pharmacology and Safety of SB-497115-GR, an Orally Active Small Molecular Weight TPO Receptor Agonist, in Chimpanzees, Rats and Dogs.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2063-2063 ◽  
Author(s):  
Teresa Sellers ◽  
Timothy Hart ◽  
Michael Semanik ◽  
Krishna Murthy
Keyword(s):  
Molecular Weight ◽  
Clinical Trials ◽  
Receptor Agonist ◽  
Fold Increase ◽  
Distinct Species ◽  
In Vivo Studies ◽  
Small Molecular Weight ◽  
Platelet Counts

Abstract SB 497115-GR is a small molecular weight Tpo receptor (TpoR) agonist that has properties similar to thrombopoietin (TPO), primarily inducing proliferation and differentiation of megakaryocytes from bone marrow progenitor cells. SB-497115-GR is being developed for the treatment of thrombocytopenias, such as immune thrombocytopenic purpura. In vitro and in vivo studies have demonstrated that SB-497115-GR has very distinct species specificity. SB-497115 or other molecules in this class induced dose dependent STAT activation in platelets from humans and chimpanzees but not in platelets from laboratory animal species commonly used in drug safety studies. In order to demonstrate in vivo activity of SB-497115-GR, a single dose and 5 daily dose pharmacology and safety study in chimpanzees was conducted. To support initiation of clinical trials, a comprehensive package of toxicology studies was conducted including studies up to 14 days duration in rats and dogs. All procedures involving the care and use of animals in these studies were reviewed and approved by the appropriate Institutional Animal Care and Use Committees. Female chimpanzees (1–3/group) were administered vehicle or SB-497115-GR at doses of 0.1 to 10 mg/kg/day by oral gavage. For toxicology studies, SB-497115-GR was administered orally to rats (10/sex/group) by gavage at doses of 3 to 40 mg/kg/day and to dogs (3/sex/group) by capsule at doses of 3 to 30 mg/kg/day for 14 days. SB-497115-GR was well tolerated in chimpanzees, rats and dogs at all doses tested. In chimpanzees, no treatment related increases in platelet counts were observed after administration of single doses of up to 10 mg/kg or 5 daily doses of up to 3 mg/kg/day. However, following 5 daily doses of 10 mg/kg/day SB-497115-GR, there was a 1.3- to 2.4-fold increase in circulating platelet counts in 3 chimpanzees. A similar change in reticulated platelet counts was observed preceding this increase. In contrast, there was no effect of treatment for up to 14 days on platelet counts in rats or dogs. In conclusion, SB-497115-GR, an orally bioavailable small molecular weight agonist of the TpoR, has been shown to increase platelet counts in chimpanzees. These in vivo data confirm the in vitro data demonstrating the unique species-specific effects of this novel Tpo receptor agonist on platelets and were predictive of a pharmacodynamic effect currently being observed in human clinical trials.

Fate of receptor-bound human chorionic gonadotrophin in pseudopregnant rat ovaries perfused in vitro

1985 ◽  
Vol 109 (1) ◽  
pp. 115-121
Author(s):  
Hannu Rajaniemi ◽  
Jan Sogn ◽  
Paul Holmes ◽  
Björn Källfelt ◽  
Per Olof Janson
Keyword(s):  
Molecular Weight ◽  
Histological Examination ◽  
Gel Filtration ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Cell Periphery ◽  
Small Molecular Weight ◽  
Immunohistochemical Studies

Abstract. Pseudopregnant rats were injected with [125I] hCG, anaesthesized 1 h later and after cannulation of the aorta the ovaries were isolated and perfused with Gey & Gey buffer containing 0.2% BSA. The release of radioactivity was monitored for 2 h and analyzed by gel filtration. Five to ten per cent of the radioactivity was released within 2 h and represented small molecular weight peptides and iodotyrosine and [125I]hCG. Analysis of the ovarian radioactivity prior to and after perfusion revealed that virtually all hCG was receptor-bound. Loading the medium with unlabelled hCG displaced [125I]hCG from the receptor but did not enhance its degradation. Histological examination showed that the ovarian tissues were still intact after the 2 h perfusion. Immunohistochemical studies revealed a localization of the hCG at the cell periphery both prior to and after perfusion. These results provide evidence showing that the rate of internalization of receptor-hCG complexes in rat luteal cells is slow in vivo.

Integrated effects of multiple modulators on human liver glycogen phosphorylase a

2002 ◽  
Vol 283 (1) ◽  
pp. E29-E37 ◽  
Author(s):  
Nacide Ercan-Fang ◽  
Mary C. Gannon ◽  
Virginia L. Rath ◽  
Judith L. Treadway ◽  
Miriam R. Taylor ◽  
...  
Keyword(s):  
Human Liver ◽  
Glycogen Phosphorylase ◽  
Hepatic Glucose Production ◽  
Active Form ◽  
Glucose Production ◽  
Liver Glycogen ◽  
Glucose Inhibition ◽  
Hepatic Glucose ◽  
Intracellular Concentrations

Hepatic glucose production is increased in people with type 2 diabetes. Glucose released from storage in liver glycogen by phosphorylase accounts for ∼50% of the glucose produced after an overnight fast. Therefore, understanding how glycogenolysis in the liver is regulated is of great importance. Toward this goal, we have determined the kinetic characteristics of recombinant human liver glycogen phosphorylase a (HLGP a) (active form) and compared them with those of the purified rat enzyme (RLGP a). The Michaelis-Menten constant ( K m) of HLGP a for Pi, 5 mM, was about fivefold greater than the K m of RLGP a. Two Pi (substrate) concentrations were used (1 and 5 mM) to cover the physiological range for Pi. Other effectors were added at estimated intracellular concentrations. When added individually, AMP stimulated, whereas ADP, ATP and glucose inhibited, activity. These results were similar to those of the RLGP a. However, glucose inhibition was about twofold more potent with the human enzyme. UDP-glucose, glucose 6-phosphate, and fructose 1-phosphate were only minor inhibitors of both enzymes. We reported previously that when all known effectors were present in combination at physiological concentrations, the net effect was no change in RLGP a activity. However, the same combination reduced HLGP a activity, and the inhibition was glucose dependent. We conclude that a combination of the known effectors of phosphorylase a activity, when present at estimated intracellular concentrations, is inhibitory. Of these effectors, only glucose changes greatly in vivo. Thus it may be the major regulator of HLGP a activity.

scholarly journals Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo

1998 ◽  
Vol 95 (4) ◽  
pp. 1776-1781 ◽  
Author(s):  
W. H. Martin ◽  
D. J. Hoover ◽  
S. J. Armento ◽  
I. A. Stock ◽  
R. K. McPherson ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Human Liver ◽  
Glycogen Phosphorylase ◽  
Liver Glycogen ◽  
Glycogen Phosphorylase Inhibitor
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