Abstract
The question of whether the effects of insulin and glucagon on hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity are mediated largely by changes in the phosphorylation state of the enzyme or by changes in the quantity of enzyme protein was investigated by measuring enzyme protein and mRNA levels. If phosphorylation/dephosphorylation is responsible for the observed changes in HMG-CoA reductase activity, one would not expect to see changes in immunoreactive protein or mRNA levels in response to induction of diabetes, administration of insulin, or administration of insulin and glucagon. It was found that hepatic HMG-CoA reductase mRNA levels were decreased to 12% of control in diabetic rats. Immunoreactive protein was reduced to essentially undetectable levels. Administration of insulin restored both mRNA and immunoreactive protein levels. Glucagon blocked these effects. Enzyme activity changes were fully accounted for by changes in HMG-CoA reductase mRNA and immunoreactive protein. Fasting caused parallel falls in HMG-CoA reductase activity and immunoreactive protein levels with a lesser effect on mRNA levels. The insulin-mediated changes in HMG-CoA reductase gene expression correlated well with changes in blood glucose levels, indicating a physiological effect. Taken together, these results indicate that insulin and glucagon regulate HMG-CoA reductase gene expression largely at the level of enzyme protein through changes in mRNA concentrations.
Highlights
Cagon on hepaticS-hydroxy-3-methylglutaryl-coenzymereductase activity suggestedthat thiseffect might be mediated A (HMG-CoA)reductase activity are mediated largbeyly by an alteration in dthegeree of phosphorylation of the enzyme
If insulin andglucagon act to alterHMG-CoA reductase ac- Effects of Diabetes and Insulin neatment-The effects of tivity largely viaphosphorylationldephosphorylationof the en- induction of diabetes and insulin treatmenton hepatic HMG
We activity levels were determineAd.s shown in Fig. 1,induction of have investigated the effects of diabetes and treatment with diabetes rapidly lowered hepatic HMG-CoA reductase mRNA
Summary
0.10 0.11 00.1.137 in diabetic rats and that administration of pharmacological line phosphatase-conjugatedsecond antibody followingthe manufacturdoses of insulin restored these levels within 2 h [22]. We showed that immunoreactive HMG-CoA reductase protein levels changed in a similar fashion [22]. These changes fully accounted for the observed changes in enzyme activity. Taken together, these observations question the physiological significance of changes in phosphorylation state mediating shorter's protocol. Enzyme Activity-HMG-CoA reductase activity was determined in liver microsomes using the thin layer chromatography procedure for isolation of product as described previously [24]. Protein concentrations were determined by a biuret method [24]. Enzyme activities are expressed in termosf nanomoles/minute/milligramof microsomal protein.
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