scholarly journals Conditions that result in the mobilization and influx of Ca2+ into rat hepatocytes induce the rapid loss of 3-hydroxy-3-methylglutaryl-CoA reductase activity that is not reversed by phosphatase treatment

1990 ◽  
Vol 269 (2) ◽  
pp. 373-379 ◽  
Author(s):  
V A Zammit ◽  
A M Caldwell
Keyword(s):  
Enzyme Activity ◽  
Okadaic Acid ◽  
Reductase Activity ◽  
Total Activity ◽  
Ionophore A23187 ◽  
Hmg Coa Reductase ◽  
Pronounced Increase ◽  
Phosphorylation State ◽  
Total Enzyme Activity ◽  
Coa Reductase

We investigated the effects of conditions that induce Ca2+ mobilization from intracellular stores and Ca2+ influx into hepatocytes on the expressed and total (fully dephosphorylated) activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Vasopressin and phenylephrine when added alone had small or negligible effects on the phosphorylation state of the enzyme, as judged from the expressed/total activity ratio. However, when added in combination with glucagon, they elicited appreciable increases in the phosphorylation of the enzyme. Glucagon on its own had no effect either on phosphorylation state or on total HMG-CoA reductase activity during 40 min of incubation. Under conditions of sustained Ca2+ influx (i.e. vasopressin or phenylephrine plus glucagon), there was a marked loss of total HMG-CoA reductase activity. This effect was more pronounced when vasopressin was used; 50% of the enzyme activity was lost within 40 min. The involvement of Ca2+ in these effects was verified directly by the use of ionophore A23187. Its addition to hepatocytes resulted both in a very pronounced increase in the phosphorylation state of the enzyme and in the loss of 50% of the total activity within 30 min. There was no correlation between the ability of any set of conditions to increase the phosphorylation of the enzyme and the subsequent loss of total HMG-CoA reductase activity. The latter parameter appeared to be directly related, however, to the maintenance of prolonged Ca2+ influx, as indicated by the continued activation of glycogen phosphorylase, measured in the same cells. The lack of a causal relationship between increased phosphorylation and loss of total activity was demonstrated directly by studies in which okadaic acid was used to induce phosphorylation of HMG-CoA reductase in hepatocytes by inhibition of phosphatase 1 and 2A activities. This was not accompanied by any loss of total enzyme activity. Neither did okadaic acid enhance the loss of reductase induced by A23187 when the two agents were added together. It is concluded that altered Ca2+ fluxes in hepatocytes in vivo, under conditions of acute or chronic stress (such as may be associated with trauma or diabetes respectively), may be involved in the regulation of the expression of HMG-CoA reductase activity through alteration of enzyme concentration in the liver.

scholarly journals Direct demonstration that increased phosphorylation of 3-hydroxy-3-methylglutaryl-CoA reductase does not increase its rate of degradation in isolated rat hepatocytes

1992 ◽  
Vol 284 (3) ◽  
pp. 901-904 ◽  
Author(s):  
V A Zammit ◽  
A M Caldwell
Keyword(s):  
Rat Hepatocytes ◽  
Total Activity ◽  
Specific Rate ◽  
Ionophore A23187 ◽  
Isolated Rat Hepatocytes ◽  
Hmg Coa Reductase ◽  
Phosphorylation State ◽  
Direct Demonstration ◽  
Coa Reductase ◽  
Isolated Rat

Increased phosphorylation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase has been suggested to target the protein towards an increased rate of degradation. Our previous observations [Zammit & Caldwell (1990) Biochem. J. 269, 373-379] suggested that, although Ca(2+)-mobilizing hormones and other effectors can alter both the phosphorylation state of the enzyme and its total activity in isolated rat hepatocytes, there appears to be no causal correlation between the two parameters. In the present paper we set out to make direct measurements of the specific rate of degradation of 35S-labelled HMG-CoA reductase in hepatocytes treated with agents that produced very marked and prolonged increases in the degree of phosphorylation of the protein, through different mechanisms. Okadaic acid (which inhibits phosphatases 1 and 2A), fructose (which increases cellular AMP through its metabolism to fructose 1-phosphate) and the Ca2+ ionophore A23187 (which also raises cellular AMP through an unknown mechanism) were all unable to alter the rate of HMG-CoA reductase degradation. We conclude that the basal rate of degradation of HMG-CoA reductase is unaffected by its phosphorylation state and that a transiently increased degree of phosphorylation cannot be the mechanism through which mevalonate increases the rate of degradation of the enzyme in rat hepatocytes and other cell types.

scholarly journals The roles of different protein kinases and of calmodulin in the effects of Ca2+ mobilization on 3-hydroxy-3-methylglutaryl-CoA reductase activity in isolated rat hepatocytes

1991 ◽  
Vol 273 (2) ◽  
pp. 485-488 ◽  
Author(s):  
V A Zammit ◽  
A M Caldwell
Keyword(s):  
Protein Kinase ◽  
Reductase Activity ◽  
Rat Hepatocytes ◽  
Total Activity ◽  
Isolated Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Hmg Coa Reductase ◽  
Lysosomal Proteolysis ◽  
Dependent Protein ◽  
Coa Reductase

The roles of protein kinase C, Ca2+/calmodulin-dependent protein kinase and AMP-activated protein kinase in the phosphorylation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase induced by Ca2(+)-mobilizing conditions in isolated hepatocytes were investigated. Only partial evidence for the involvement of AMP-activated kinase was found. Antagonism of calmodulin action prolonged the decrease in expressed/total activity ratio induced by vasopressin plus glucagon. Protease inhibitors active against Ca2(+)-dependent cytosolic proteases or lysosomal proteolysis did not attenuate the loss of total HMG-CoA reductase induced by glucagon plus vasopressin, but calmodulin antagonists largely prevented this effect.

5α-Reductase activity in stroma and epithelium of rat prostate and epididymis. A contribution to elucidation of the mechanism for development of hyperplastic growth of prostatic tissue

1983 ◽  
Vol 103 (2) ◽  
pp. 273-281 ◽  
Author(s):  
Ole Djøseland ◽  
Nicholas Bruchovsky ◽  
Paul S. Rennie ◽  
Navdeep Otal ◽  
Sian Høglo
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Reductase Activity ◽  
Major Change ◽  
Growth Stimulation ◽  
Total Activity ◽  
Prostatic Tissue ◽  
Ventral Prostate ◽  
Total Enzyme Activity ◽  
Abnormal Growth

Abstract. The 5α-reductase activity was assayed in homogenates of stroma and epithelium in the rat ventral prostate and epididymis. Samples consisting of 0.3 mg/ml tissue protein in TES buffer, pH 7.0 were incubated at 37°C for 30 min in the presence of 50 nm [1,2-3H]testosterone and a NADPH-generating system started with 5 × 10−4 m NADP. The yield of 5α-reduced metabolites, as established by using thin-layer chromatography, gave an estimate of enzyme activity. Whereas the specific activity of 5α-reductase was highest in prostatic stroma and epididymal epithelium, most of the total enzyme activity was associated with the epithelium in both the prostate and epididymis. The effect of dihydrotestosterone on specific activity of 5α-reductase was studied by administering the hormone to 7-day castrated rats. In prostate, the specific activity of both stromal and epithelium forms of the enzyme reached a maximum after 4 days of treatment. In epididymis only the epithelial form of 5α-reductase underwent a major change in specific activity, the latter peaking after 8–12 days of treatment. Furthermore, while the total activity of 5α-reductase in the prostatic tissue fractions could be induced by as much as 4-fold the normal control values, the epididymal enzyme could not be induced above the normal level either in the stroma or the epithelium. This may explain the relative resistance of epididymis to abnormal growth stimulation under the influence of hormones.

scholarly journals Role of reversible phosphorylation in mediating changes in the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase during pregnancy and lactation in the rat

1987 ◽  
Vol 248 (3) ◽  
pp. 993-996 ◽  
Author(s):  
R A Easom ◽  
V A Zammit
Keyword(s):  
Reductase Activity ◽  
Post Partum ◽  
Active Form ◽  
Total Activity ◽  
Female Rats ◽  
Hmg Coa Reductase ◽  
Pregnancy And Lactation ◽  
Coa Reductase

1. The expressed and total (completely dephosphorylated) activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase were measured in microsomal fractions isolated from cold-clamped liver samples from female rats in various stages of the reproductive cycle. 2. There was little change in total HMG-CoA reductase activity during pregnancy and early lactation, but after 2 days post partum there was a marked increase in total activity. 3. The expressed/total activity ratio of HMG-CoA reductase showed a profound decrease during the last 2 days of pregnancy. The fraction of the enzyme in the active form increased progressively during the first 2 days of lactation. 4. The combined effect of these changes was that the expressed activity of HMG-CoA reductase changed in parallel with the known changes in the hepatic rate of cholesterogenesis during pregnancy and lactation in vivo.

scholarly journals Diurnal variation in the fraction of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the active form in the mammary gland of the lactating rat

1986 ◽  
Vol 239 (2) ◽  
pp. 285-293 ◽  
Author(s):  
R A Smith ◽  
B Middleton ◽  
D W West
Keyword(s):  
Reductase Activity ◽  
Active Form ◽  
Total Activity ◽  
Maximum Activity ◽  
Covalent Modification ◽  
Light Phase ◽  
Hmg Coa Reductase ◽  
Enzyme Molecules ◽  
Coa Reductase ◽  
Homogenization Medium

‘Expressed’ and ‘total’ activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in freeze-clamped samples of mammary glands from lactating rats at intervals throughout the 24 h light/dark cycle. ‘Expressed’ activities were measured in microsomal fractions isolated and assayed in the presence of 100 mM-KF. ‘Total’ activities were determined in microsomal preparations from the same homogenates but washed free of KF and incubated with exogenously added sheep liver phosphoprotein phosphatase before assay. Both ‘expressed’ and ‘total’ activities of HMG-CoA reductase underwent a diurnal cycle, which had a major peak 6 h into the light phase and a nadir 15 h later, i.e. 9 h into the dark period. Both activities showed a secondary peak of activity (around 68% of the maximum activity) at the time of changeover from dark to light, with a trough in the value of the ‘expressed’ activity that was close to the nadir value. ‘Expressed’ activity was lower than ‘total’ at all time points, indicating the presence of enzyme molecules inactivated by covalent phosphorylation. Nevertheless the ‘expressed’/‘total’ activity ratio was comparatively constant and varied only between 43% and 75%. Immunotitration of enzyme activity, with antiserum raised in sheep against purified rat liver HMG-CoA reductase, confirmed the presence of both active and inactive forms of the enzyme and indicated that at the peak and nadir the variation in ‘expressed’ HMG-CoA reductase activity resulted from changes in the total number of enzyme molecules rather than from covalent modification. The sample obtained after 3 h of the light phase exhibited an anomalously low ‘total’ HMG-CoA reductase activity, which could be increased when Cl- replaced F- in the homogenization medium. The result suggests that at that time the activity of the enzyme could be regulated by mechanisms other than covalent phosphorylation or degradation.

Cholesterol-mediated regulation of HMG-CoA reductase in microsomes from human skin fibroblasts and rat liver

1990 ◽  
Vol 68 (3) ◽  
pp. 674-679 ◽  
Author(s):  
R. George ◽  
P. J. Davis ◽  
L. Luong ◽  
M. J. Poznansky
Keyword(s):  
Tissue Culture ◽  
Enzyme Activity ◽  
Human Skin ◽  
Rat Liver ◽  
Reductase Activity ◽  
Cholesterol Content ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Hmg Coa Reductase ◽  
Coa Reductase

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was determined in microsomes from human skin fibroblasts and rat liver that had been variously manipulated in vivo or in tissue culture to up- and down-regulate the enzyme. The cholesterol content of these microsomal preparations was then altered by depletion to or enrichment from either cholesterol-free or cholesterol-rich lipid vesicles. Microsomes from human skin fibroblasts responded to cholesterol depletion by increasing HMG-CoA reductase activity and by decreasing it in response to cholesterol enrichment. This was independent of the initial enzyme activity or the tissue culture conditions. Alterations in cholesterol content of rat liver microsomes in vitro failed to demonstrate any significant changes in HMG-CoA reductase activity whether the microsomes started with low enzyme activity (cholesterol-fed rats) or with high enzyme activity (cholestyramine-treated rats). The results are discussed in relation to previously published data and in respect to differences in the control of the human skin fibroblast and rat liver enzymes.Key words: cholesterol, HMG-CoA reductase, microsomes, fibroblasts, rat liver.

scholarly journals Effects of diabetes on the expressed and total activities of 3-hydroxy-3-methylglutaryl-CoA reductase in rat liver in vivo. Reversal by insulin treatment

1985 ◽  
Vol 230 (3) ◽  
pp. 747-752 ◽  
Author(s):  
R A Easom ◽  
V A Zammit
Keyword(s):  
Activity Ratio ◽  
Reductase Activity ◽  
Active Form ◽  
Fold Increase ◽  
Total Activity ◽  
Diabetic Rats ◽  
Diurnal Rhythms ◽  
Hmg Coa Reductase ◽  
Coa Reductase

The expressed and total activities of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase (EC 1.1.1.34) were measured in microsomal fractions prepared from cold-clamped liver samples [Easom & Zammit (1984) Biochem. J. 220, 733-738] from control or insulin-treated diabetic animals. Streptozotocin-induced diabetes resulted in a marked decrease in total activity of HMG-CoA reductase and in the fraction of the enzyme in the active form, but appreciable effects were only observed in the liver of animals in which the blood glucose was above 20 mM. Intravenous infusion of insulin into diabetic rats resulted in a rapid (less than 20 min) and total dephosphorylation of the enzyme in vivo without any change in total activity. Longer-term (4 h) treatment with insulin (injected intraperitoneally) produced a rapid increase in expressed/total HMG-CoA reductase activity ratio to about 90%, followed, after a lag of 2-3 h, by a 5-6-fold increase in total activity. These observations are discussed with respect to the possible role of insulin in generating and maintaining the respective diurnal rhythms in total and in expressed/total HMG-CoA reductase activity ratio observed for normal animals in vivo [Easom & Zammit (1984) Biochem. J. 220, 739-745].

scholarly journals Acute effects of starvation and treatment of rats with anti-insulin serum, glucagon and catecholamines on the state of phosphorylation of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase in vivo

1987 ◽  
Vol 241 (1) ◽  
pp. 183-188 ◽  
Author(s):  
R A Easom ◽  
V A Zammit
Keyword(s):  
Intravenous Injection ◽  
Activity Ratio ◽  
Reductase Activity ◽  
Active Form ◽  
Fold Increase ◽  
Total Activity ◽  
Dark Phase ◽  
Hmg Coa Reductase ◽  
Coa Reductase

The fraction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in the dephosphorylated (active) form in rat liver in vivo was measured after various experimental treatments of animals. Intraperitoneal injection of glucose (to raise serum insulin concentrations) into rats 4 h into the light phase (L-4) resulted in a transient (30 min) increase in the expressed (E)/total (T) activity ratio of HMG-CoA reductase without any change in total activity (obtained after complete dephosphorylation of the enzyme). Conversely, intravenous injection of guinea-pig anti-insulin serum into rats 4 h into the dark phase (D-4) significantly depressed the E/T ratio within 20 min. Intravenous injection of glucagon into normal rats at this time point did not affect the degree of phosphorylation of the enzyme, in spite of a 10-fold increase in hepatic cyclic AMP concentration induced by the hormone treatment. A 3-fold increase in the concentration of the cyclic nucleotide induced by adrenaline infusion was similarly ineffective in inducing any change in expressed or total activities of hepatic HMG-CoA reductase. However, when insulin secretion was inhibited, either by the induction of streptozotocin-diabetes or by simultaneous infusion of somatostatin, glucagon treatment was able to depress the expressed activity of HMG-CoA reductase (i.e. it increased the phosphorylation of the enzyme). Therefore insulin appears to have a dominant role in the regulation of the phosphorylation state of hepatic HMG-CoA reductase. In apparent corroboration of this suggestion, short-term 4 h food deprivation of animals before D-4 resulted in a marked decrease in the E/T activity ratio of reductase, which was not affected further by an additional 8 h starvation. By contrast, the total activity of the enzyme was not significantly affected by 4 h starvation, but was markedly diminished after 12 or 24 h starvation. Longer-term starvation also produced a chronic increase in the degree of phosphorylation of the enzyme. These results are discussed in relation to the role of reversible phosphorylation in the control of hepatic HMG-CoA reductase activity in vivo.

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