Colosia A D, Marker A J, Lange A J, el-Maghrabi M R, Granner D K, Tauler A, Pilkis J, Pilkis S J
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232.
J Biol Chem. 1988 Dec 15;263(35):18669-77.
The effects of fasting/refeeding and untreated or insulin-treated diabetes on the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its mRNA in rat liver were determined. Both enzymatic activities fell to 20% of control values with fasting or streptozotocin-induced diabetes and were coordinately restored to normal within 48 h of refeeding or 24 h of insulin administration. These alterations in enzymatic activities were always mirrored by corresponding changes in amount of enzyme as determined by phosphoenzyme formation and immunoblotting. In contrast, mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase did not decrease during starvation or in diabetes, but there was a 3-6-fold increase upon refeeding a high carbohydrate diet to starved rats or insulin treatment of diabetic rats. The decrease of the enzyme in starved or diabetic rats without associated changes in mRNA levels suggests a decrease in the rate of mRNA translation, an increase in enzyme degradation, or both. The rise in enzyme amount and mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with refeeding and insulin treatment suggests an insulin-dependent stimulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression. Northern blots of RNA from heart, brain, kidney, and skeletal muscle probed with restriction fragments of a full-length cDNA from liver showed that only skeletal muscle contained an RNA species that hybridized to any of the probes. Skeletal muscle mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was 2.0 kilobase pairs but in contrast to the liver message (2.2 kilobase pairs) was not regulated by refeeding.
研究了禁食/再喂养以及未治疗或胰岛素治疗的糖尿病对大鼠肝脏中双功能酶6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶及其mRNA的影响。禁食或链脲佐菌素诱导的糖尿病会使两种酶活性降至对照值的20%，再喂养48小时或给予胰岛素24小时后，酶活性会协同恢复正常。通过磷酸化酶形成和免疫印迹法测定，这些酶活性的改变总是伴随着酶量的相应变化。相比之下，6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶的mRNA在饥饿或糖尿病期间并未减少，但饥饿大鼠重新喂食高碳水化合物饮食或糖尿病大鼠接受胰岛素治疗后，其mRNA会增加3至6倍。饥饿或糖尿病大鼠中酶的减少而mRNA水平无相关变化，提示mRNA翻译速率降低、酶降解增加或两者皆有。再喂养和胰岛素治疗后6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶的酶量和mRNA增加，提示胰岛素依赖性刺激6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶基因表达。用来自肝脏的全长cDNA的限制性片段探测心脏、大脑、肾脏和骨骼肌的RNA进行Northern印迹分析，结果显示只有骨骼肌含有与任何一种探针杂交的RNA物种。骨骼肌中6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶的mRNA为2.0千碱基对，但与肝脏中的mRNA（2.2千碱基对）不同，其不受再喂养的调节。
