Ranganathan G, Li C, Kern P A
Department of Medicine, Division of Endocrinology, University of Arkansas for Medical Sciences and The Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205, USA.
J Biol Chem. 2000 Dec 29;275(52):40986-91. doi: 10.1074/jbc.M008775200.
Adipose tissue lipoprotein lipase (LPL) activity is decreased in patients with poorly controlled diabetes, and this contributes to the dyslipidemia of diabetes. To study the mechanism of this decrease in LPL, we studied adipose tissue LPL expression in male rats with streptozotocin-induced diabetes. Heparin releasable and extractable LPL activity in the epididymal fat decreased by 75-80% in the diabetic group and treatment of the rats with insulin prior to sacrifice reversed this effect. Northern blot analysis indicated no corresponding change in LPL mRNA levels. However, LPL synthetic rate, measured using [(35)S]methionine pulse labeling, was decreased by 75% in the diabetic adipocytes, and insulin treatment reversed this effect. These results suggested regulation of LPL at the level of translation. Diabetic adipocytes demonstrated no change in the distribution of LPL mRNA associated with polysomes, suggesting no inhibition of translation initiation. Addition of cytoplasmic extracts from control and diabetic adipocytes to a reticulocyte lysate system demonstrated the inhibition of LPL translation in vitro. Using different LPL mRNA transcripts in this in vitro translation assay, we found that the 3'-untranslated region (UTR) of the LPL mRNA was important in controlling translation inhibition by the cytoplasmic extracts. To identify the specific region involved, gel shift analysis was performed. A specific shift in mobility was observed when diabetic cytoplasmic extract was added to a transcript containing nucleotides 1818-2000 of the LPL 3'-UTR. Thus, inhibition of translation is the predominant mechanism for the decreased adipose tissue LPL in this insulin-deficient model of diabetes. Translation inhibition involves the interaction of a cytoplasmic factor, probably an RNA-binding protein, with specific sequences of the LPL 3'-UTR.
糖尿病控制不佳患者的脂肪组织脂蛋白脂肪酶（LPL）活性降低，这导致了糖尿病的血脂异常。为研究LPL活性降低的机制，我们研究了链脲佐菌素诱导的糖尿病雄性大鼠的脂肪组织LPL表达。糖尿病组附睾脂肪中肝素可释放和可提取的LPL活性降低了75 - 80%，在处死大鼠前用胰岛素治疗可逆转此效应。Northern印迹分析表明LPL mRNA水平无相应变化。然而，使用[³⁵S]甲硫氨酸脉冲标记法测得的糖尿病脂肪细胞中LPL合成率降低了75%，胰岛素治疗可逆转此效应。这些结果提示LPL在翻译水平受到调控。糖尿病脂肪细胞中与多核糖体相关的LPL mRNA分布无变化，提示翻译起始未受抑制。将对照和糖尿病脂肪细胞的细胞质提取物添加到网织红细胞裂解物系统中，证明了体外LPL翻译受到抑制。在该体外翻译试验中使用不同的LPL mRNA转录本，我们发现LPL mRNA的3'非翻译区（UTR）在控制细胞质提取物对翻译的抑制中起重要作用。为确定所涉及的特定区域，进行了凝胶迁移分析。当将糖尿病细胞质提取物添加到包含LPL 3'-UTR核苷酸1818 - 2000的转录本中时，观察到迁移率有特定变化。因此，在这种胰岛素缺乏的糖尿病模型中，翻译抑制是脂肪组织LPL降低的主要机制。翻译抑制涉及一种细胞质因子（可能是一种RNA结合蛋白）与LPL 3'-UTR的特定序列相互作用。
