PI3-kinase activity modulates apo B available for hepatic VLDL production in apobec-1−/− mice

2006 ◽  
Vol 291 (3) ◽  
pp. G382-G388 ◽  
Author(s):  
Doru V. Chirieac ◽  
Nicholas O. Davidson ◽  
Charles E. Sparks ◽  
Janet D. Sparks
Keyword(s):  
Insulin Resistance ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Apo B ◽  
Glucose Injection ◽  
Primary Mouse ◽  
Mouse Hepatocytes ◽  
Vldl Triglyceride

Insulin regulates hepatic VLDL production by activation of phosphatidylinositide 3-kinase (PI3-kinase) which decreases apo B available for lipid assembly. The current study evaluated the dependence of the VLDL apolipoprotein B (apo B) pathway on PI3-kinase activity in vivo. VLDL production was examined in B100 only, apo B mRNA editing catalytic subunit 1 ( apobec-1 −/−) mice, using the Triton WR 1339 method. Glucose injection suppressed VLDL triglyceride production by 28% in male and by 32% in female mice compared with saline-injected controls. When wortmannin was injected to inhibit PI3-kinase, VLDL triglyceride production was increased by 52% in males and by 89% in females, and VLDL B100 levels paralleled triglyceride changes. Pulse-chase experiments in primary mouse hepatocytes showed that wortmannin increased net freshly synthesized B100 availability by >35%. To test whether physiological insulin resistance produced equivalent effects to wortmannin, we studied male apobec-1 −/− mice who became hyperlipidemic on being fed a fructose-enriched diet. Fructose-fed apobec-1 −/− mice had significantly higher VLDL triglyceride and B100 production rates compared with chow-fed mice, and rates were refractile to glucose or wortmannin. Hepatic VLDL triglyceride and B100 production in wortmannin-injected chow-fed mice equaled that observed in fructose-fed mice. Together, results suggest in vivo and in vitro that wortmannin-sensitive PI3-kinases maintain a basal level of VLDL suppression that is sensitive to changes in activation and that can increase VLDL production when PI3-kinase is inhibited to levels similar to those induced by insulin resistance.

scholarly journals Crosstalk between the AMP-activated kinase and insulin signaling pathways rescues murine blastocyst cells from insulin resistance

Reproduction ◽  
2008 ◽  
Vol 136 (3) ◽  
pp. 335-344 ◽  
Author(s):  
Erica Louden ◽  
Maggie M Chi ◽  
Kelle H Moley
Keyword(s):  
Insulin Resistance ◽  
Signaling Pathways ◽  
Insulin Signaling ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Ampk Activation ◽  
Embryonic Cells ◽  
Insulin Resistant

Maternal insulin resistance results in poor pregnancy outcomes. In vivo and in vitro exposure of the murine blastocyst to high insulin or IGF1 results in the down-regulation of the IGF1 receptor (IGF1R). This in turn leads to decreased glucose uptake, increased apoptosis, as well as pregnancy resorption and growth restriction. Recent studies have shown that blastocyst activation of AMP-activated protein kinase (AMPK) reverses these detrimental effects; however, the mechanism was not clear. The objective of this study was to determine how AMPK activation rescues the insulin-resistant blastocyst. Using trophoblast stem (TS) cells derived from the blastocyst, insulin resistance was recreated by transfecting with siRNA to Igf1r and down-regulating expression of the protein. These cells were then exposed to AMPK activators 5-aminoimidazole-4-carboxamide riboside and phenformin, and evaluated for apoptosis, insulin-stimulated 2-deoxyglucose uptake, PI3-kinase activity, and levels of phospho-AKT, phospho-mTor, and phospho-70S6K. Surprisingly, disrupted insulin signaling led to decreased AMPK activity in TS cells. Activators reversed these effects by increasing the AMP/ATP ratio. Moreover, this treatment increased insulin-stimulated 2-deoxyglucose transport and cell survival, and led to an increase in PI3-kinase activity, as well as increased P-mTOR and p70S6K levels. This study is the first to demonstrate significant crosstalk between the AMPK and insulin signaling pathways in embryonic cells, specifically the enhanced response of PI3K/AKT/mTOR to AMPK activation. Decreased insulin signaling also resulted in decreased AMPK activation. These findings provide mechanistic targets in the AMPK signaling pathway that may be essential for improved pregnancy success in insulin-resistant states.

scholarly journals Loss of PI3 kinase association improves the sensitivity of secondary mutation of KIT to Imatinib

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Guangrong Zhu ◽  
Jun Shi ◽  
Shaoting Zhang ◽  
Yue Guo ◽  
Ling Huang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Targeted Therapy ◽  
Kinase Activity ◽  
Cell Transformation ◽  
Pi3 Kinase ◽  
Driver Mutations ◽  
Secondary Mutation ◽  
Lipid Kinase

Abstract Background KIT mutations are the predominant driver mutations in gastrointestinal stromal tumors (GISTs), and targeted therapy against KIT has improved treatment outcome dramatically. However, gaining secondary mutation of KIT confers drug resistance of GISTs leading to treatment failure. Results In this study, we found that secondary mutation of KIT dramatically increases the ligand-independent activation of the receptor and their resistance to the often used KIT inhibitor Imatinib in the treatment of GISTs. PI3 kinase plays essential roles in the cell transformation mediated by the primary mutation of KIT. We found that loss of PI3 kinase association, but not the inhibition of the lipid kinase activity of PI3 kinase, inhibits the ligand-independent activation of secondary mutations of KIT, and increases their sensitivity to Imatinib, and loss of PI3 kinase association inhibits secondary mutations of KIT mediated cell survival and proliferation in vitro. The in vivo assay further showed that the growth of tumors carrying secondary mutations of KIT is more sensitive to Imatinib when PI3 kinase association is blocked while inhibition of the lipid kinase activity of PI3 kinase cannot inhibit tumor growth, indicating that PI3 kinase is important for the drug resistance of secondary mutation of KIT independent of the lipid kinase activity of PI3 kinase. Conclusions Our results suggested that PI3 kinase is necessary for the ligand-independent activation of secondary mutations of KIT, and loss of PI3 kinase association improves the sensitivity of secondary mutations to the targeted therapy independent of the lipid kinase activity of PI3 kinase.

Antiapoptotic effect of EGF on mouse hepatocytes associated with downregulation of proapoptotic Bid protein

2003 ◽  
Vol 285 (2) ◽  
pp. G298-G308 ◽  
Author(s):  
Chantal Éthier ◽  
Valérie-Ann Raymond ◽  
Lina Musallam ◽  
Robert Houle ◽  
Marc Bilodeau
Keyword(s):  
Growth Factors ◽  
Egf Receptor ◽  
Specific Inhibitor ◽  
Pi3 Kinase ◽  
Hepatocyte Apoptosis ◽  
Antiapoptotic Effect ◽  
Primary Mouse ◽  
Major Player ◽  
Mouse Hepatocytes

Growth factors have been shown to protect cells from a variety of apoptotic stimuli. In the liver, the Fas system is thought to be very important in the genesis of hepatocyte apoptosis. Others have already shown the importance of the phosphatidylinositol 3-kinase (PI3-kinase) pathway and of increased Bcl-xl expression in the antiapoptotic effect of growth factors on hepatocytes. We investigated the effect of EGF on Bid, a BH3-only member of the Bcl-2 family and a major player in the transduction of the Fas apoptotic signal. Hepatocyte apoptosis was induced in vitro with a purified anti-mouse Fas antibody. The effect of EGF on Bid protein expression was studied on those cultures. EGF dose dependently reduced the expression of Bid protein in primary mouse hepatocyte cultures independently of Fas stimulation. This decrease was not the result of the degradation of Bid into its active p15 fragment. Treating cells with a specific inhibitor of the EGF receptor autophosphorylation completely abolished the decrease in Bid expression afforded by EGF. Treatment with LY-294002, a PI3-kinase blocker, partly reverted the effect of EGF. When apoptosis was induced in Bid-deficient hepatocytes, EGF lost its capacity to protect cells against this type of cell death. These results show that EGF decreases the expression of Bid protein and suggest that the effect of EGF on Bid is one of the mechanisms of the antiapoptotic effect of EGF.

scholarly journals Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss

2003 ◽  
Vol 162 (4) ◽  
pp. 551-563 ◽  
Author(s):  
Beth A.A. Weaver ◽  
Zahid Q. Bonday ◽  
Frances R. Putkey ◽  
Geert J.P.L. Kops ◽  
Alain D. Silk ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Cell Cycle Control ◽  
Mitotic Checkpoint ◽  
Chromosome Loss ◽  
Single Chromosome ◽  
Binding Partner ◽  
Anaphase Onset ◽  
Primary Mouse

Centromere-associated protein-E (CENP-E) is an essential mitotic kinesin that is required for efficient, stable microtubule capture at kinetochores. It also directly binds to BubR1, a kinetochore-associated kinase implicated in the mitotic checkpoint, the major cell cycle control pathway in which unattached kinetochores prevent anaphase onset. Here, we show that single unattached kinetochores depleted of CENP-E cannot block entry into anaphase, resulting in aneuploidy in 25% of divisions in primary mouse fibroblasts in vitro and in 95% of regenerating hepatocytes in vivo. Without CENP-E, diminished levels of BubR1 are recruited to kinetochores and BubR1 kinase activity remains at basal levels. CENP-E binds to and directly stimulates the kinase activity of purified BubR1 in vitro. Thus, CENP-E is required for enhancing recruitment of its binding partner BubR1 to each unattached kinetochore and for stimulating BubR1 kinase activity, implicating it as an essential amplifier of a basal mitotic checkpoint signal.

scholarly journals Glycerol induces G6pc in primary mouse hepatocytes and is the preferred substrate for gluconeogenesis both in vitro and in vivo

2019 ◽  
Vol 294 (48) ◽  
pp. 18017-18028 ◽  
Author(s):  
Katarzyna M. Kalemba ◽  
Yujue Wang ◽  
Huiting Xu ◽  
Eric Chiles ◽  
Sara M. McMillin ◽  
...  
Keyword(s):  
Primary Mouse Hepatocytes ◽  
Primary Mouse ◽  
Mouse Hepatocytes

scholarly journals Long-Term Engineered Cultures of Primary Mouse Hepatocytes for Strain and Species Comparison Studies During Drug Development

2019 ◽  
Vol 19 (3) ◽  
pp. 199-214
Author(s):  
Brenton R. Ware ◽  
Grace E. Brown ◽  
Valerie Y. Soldatow ◽  
Edward L. LeCluyse ◽  
Salman R. Khetani
Keyword(s):  
Mouse Genetics ◽  
Human Populations ◽  
Urea Synthesis ◽  
Cyp Induction ◽  
Compound Libraries ◽  
Primary Mouse Hepatocytes ◽  
Primary Mouse ◽  
Mouse Hepatocytes

Testing drugs in isogenic rodent strains to satisfy regulatory requirements is insufficient for derisking organ toxicity in genetically diverse human populations; in contrast, advances in mouse genetics can help mitigate these limitations. Compared to the expensive and slower in vivo testing, in vitro cultures enable the testing of large compound libraries toward prioritizing lead compounds and selecting an animal model with human-like response to a compound. In the case of the liver, a leading cause of drug attrition, isolated primary mouse hepatocytes (PMHs) rapidly decline in function within current culture platforms, which restricts their use for assessing the effects of longer-term compound exposure. Here we addressed this challenge by fabricating mouse micropatterned cocultures (mMPCC) containing PMHs and 3T3-J2 murine embryonic fibroblasts that displayed 4 weeks of functions; mMPCCs created from either C57Bl/6J or CD-1 PMHs outperformed collagen/Matrigel™ sandwich-cultured hepatocyte monocultures by ∼143-fold, 413-fold, and 10-fold for albumin secretion, urea synthesis, and cytochrome P450 activities, respectively. Such functional longevity of mMPCCs enabled in vivo relevant comparisons across strains for CYP induction and hepatotoxicity following exposure to 14 compounds with subsequent comparison to responses in primary human hepatocytes (PHHs). In conclusion, mMPCCs display high levels of major liver functions for several weeks and can be used to assess strain- and species-specific compound effects when used in conjunction with responses in PHHs. Ultimately, mMPCCs can be used to leverage the power of mouse genetics for characterizing subpopulations sensitive to compounds, characterizing the degree of interindividual variability, and elucidating genetic determinants of severe hepatotoxicity in humans.

Insulin-sensitive tyrosine kinase: relationship with in vivo insulin action in humans

1990 ◽  
Vol 258 (6) ◽  
pp. E964-E974
Author(s):  
B. L. Nyomba ◽  
V. M. Ossowski ◽  
C. Bogardus ◽  
D. M. Mott
Keyword(s):  
Insulin Resistance ◽  
Tyrosine Kinase ◽  
Low Dose ◽  
Kinase Activity ◽  
Receptor Kinase ◽  
Tyrosine Kinase Activity ◽  
Kinase Activation ◽  
Insulin Receptor Kinase

To investigate the relationship of insulin receptor kinase with insulin resistance in humans, we studied insulin-sensitive tyrosine kinase activity in muscle biopsies taken from 20 Pima Indians [14 nondiabetics, 6 with non-insulin-dependent mellitus (NIDDM)] during euglycemic clamps, at insulin concentrations of approximately 68 microU/ml (low dose) and approximately 1,170 microU/ml (high dose). In the nondiabetics, the low dose, insulin-induced kinase activation in vivo was 1.5-fold the activity in the fasting state (P less than 0.05), whereas in the diabetics, the kinase activity actually decreased by 40% relative to fasting (P less than 0.05). The difference in delta-kinase in vivo was significant (P less than 0.01) between the two groups. Similarly, the kinase activation in vitro in response to 1 nM insulin was lower in diabetic subjects compared with nondiabetics (P less than 0.01). These data indicate that, in NIDDM, both in vitro and in vivo insulin-stimulated tyrosine kinase activity is impaired. Among nondiabetics, the kinase sensitivity to insulin, calculated as the ratio of the kinase activity at 1 nM insulin in vitro to the kinase activity at 100 nM insulin, was positively correlated with plasma insulin concentrations 2 h after an oral glucose load (r = 0.69, P less than 0.01). Thus, in nondiabetic subjects with insulin resistance, insulin activation of the kinase is not reduced, but the kinase sensitivity to insulin increases with increasing plasma insulin levels. Therefore, the site of insulin resistance in nondiabetic subjects is distal to the insulin receptor kinase. Furthermore, it is possible that circulating insulin, by increasing the kinase sensitivity to insulin, is a determinant of the receptor kinase activity.

Effect of a high-fat-sucrose diet on in vivo insulin receptor kinase activation

1990 ◽  
Vol 259 (1) ◽  
pp. E111-E116 ◽  
Author(s):  
J. J. Boyd ◽  
I. Contreras ◽  
M. Kern ◽  
E. B. Tapscott ◽  
D. L. Downes ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Glucose Uptake ◽  
Kinase Activity ◽  
Insulin Binding ◽  
Receptor Kinase ◽  
High Fat ◽  
Glucose Injection ◽  
Insulin Receptor Kinase

Insulin-stimulated glucose uptake into muscle is depressed by high-fat-sucrose (HFS) feeding of rats. To investigate the mechanism of this insulin resistance, the in vivo activation of the insulin receptor kinase in liver and muscle of control and HFS-fed rats was determined. Rats were injected with glucose and insulin and killed 0, 5, 15, and 30 min after injection. Insulin binding was not changed in partially purified receptors from muscle of HFS rats. In control rats insulin receptor kinase activity was maximally stimulated threefold in liver at 5 min and fourfold in muscle at 15 min after insulin-glucose injection. The insulin-stimulated tyrosine kinase activity of receptors isolated from the liver of rats fed the HFS diet was decreased by 30% in comparison with the controls. In contrast, receptors isolated from muscle did not show any difference in basal or insulin-stimulated kinase activity between HFS-fed and control rats. Decreased in vivo activation of the insulin receptor kinase may be at least partially responsible for insulin resistance in liver. Because insulin binding and insulin stimulation of receptor kinase were normal in muscle of HFS-fed animals, it is concluded that the insulin resistance of glucose uptake into muscle is caused by a defect distal to the insulin receptor.

HEPATOCYTE APOPTOSIS INDUCTION BY ACETAMINOPHEN THROUGH MODULATION OF CASPASE/BAX PATHWAY IN MICE

2020 ◽  
pp. 47-52
Author(s):  
RASHA N. ABU-AJAMIEH ◽  
BAYAN Y. GHANIM ◽  
OMAR S. GAMMOH ◽  
NIDAL A. QINNA
Keyword(s):  
Vitamin C ◽  
Caspase 3 ◽  
Annexin V ◽  
Toxic Dose ◽  
Primary Mouse ◽  
Mouse Hepatocytes ◽  
Apap Hepatotoxicity ◽  
Vit C

Objective: Acetaminophen (APAP) overdose contributes to liver damage through modulation of pro-apoptotic processing. This study evaluated the involvement of caspase/Bax factors in APAP hepatotoxicity in vivo and in vitro. Methods: The involvement of caspase/Bax factors in APAP hepatotoxicity was evaluated in BALB/c mice and on isolated primary mouse hepatocytes. In vitro MTT assay was carried out on primary cultured mouse hepatocytes treated with APAP (2.5, 5, 10 mmol) and Annexin V/PI staining was employed to cell suspension for imaging under fluorescence microscopy. In addition, caspase-3 concentrations were determined in cell lysates. In vivo, mice were treated with a toxic dose of APAP (700 mg/kg) and immunodetection of Bax was made by Western Blot. Vitamin C (Vit C) was used as a hepato-protectant due to its known antioxidant activities. Results: In vitro dose-dependent increase in mitochondrial electron transport capacity was evident in isolated mouse primary hepatocytes incubated with the high dose of APAP (10 mmol) compared to both nontreated cells and cells pre-treated with Vitamin C (Vit C) (0.5 mmol) (p<0.05). Apoptosis was confirmed in hepatocytes through Annexin V staining after APAP treatment and the signal was reduced when hepatocytes were pre-treated with Vit C. In addition, caspase-3 concentration was decreased in cells pretreated with Vit C prior to APAP exposure. In vivo, Bax immunodetection utilizing western blotting was increased in mice treated with the toxic dose of APAP (700 mg/kg) and attenuated through pre-treatment with Vit C. Conclusion: Modulation of apoptotic caspase/Bax pathway is present in hepatocytes undergoing APAP-induced toxicity.

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