Abstract
During short term fasting, lipoprotein lipase (LPL) activity in rat adipose tissue is rapidly down-regulated. This down-regulation occurs on a posttranslational level; it is not accompanied by changes in LPL mRNA or protein levels. The LPL activity can be restored within 4 h by refeeding. Previously, we showed that during fasting there is a shift in the distribution of lipase protein toward an inactive form with low heparin affinity. To study the nature of the regulatory mechanism, we determined the in vivo turnover of LPL activity, protein mass, and mRNA in rat adipose tissue. When protein synthesis was inhibited with cycloheximide, LPL activity and protein mass decreased rapidly and in parallel with half-lives of around 2 h, and the effect of refeeding was blocked. This indicates that maintaining high levels of LPL activity requires continuous synthesis of new enzyme protein. When transcription was inhibited by actinomycin, LPL mRNA decreased with half-lives of 13.3 and 16.8 h in the fed and fasted states, respectively, demonstrating slow turnover of the LPL transcript. Surprisingly, when actinomycin was given to fed rats, LPL activity was not down-regulated during fasting, indicating that actinomycin interferes with the transcription of a gene that blocks the activation of newly synthesized LPL protein. When actinomycin was given to fasted rats, LPL activity increased 4-fold within 6 h, even in the absence of refeeding. The same effect was seen with alpha-amanitin, another inhibitor of transcription. The response to actinomycin was much less pronounced in aging rats, which are obese and insulin-resistant. These data suggest a default state where LPL protein is synthesized on a relatively stable mRNA and is processed into its active form. During fasting, a gene is switched on whose product prevents the enzyme from becoming active even though synthesis of LPL protein continues unabated.
Highlights
Lipoprotein lipase (LPL)1 plays an important physiological role in regulating the release of fatty acids from triglyceriderich lipoproteins [1,2,3,4]
lipoprotein lipase (LPL) activity and protein mass dropped to 40 –50% of control within 2 h after the administration of cycloheximide and decreased more slowly, reaching 20% of control at 6 h. These data demonstrate that after the block of protein synthesis by cycloheximide, the half-lives of LPL activity and protein mass in adipose tissue are less than 2 h and that LPL activity decreases as a result of protein turnover and not because the enzyme protein loses its catalytic activity
In the ␣-amanitin-treated group, LPL specific activity increased (0.35 milliunit/mg, p Ͻ 0.001 versus fasted controls, data not shown). These results indicate that the effect of actinomycin on LPL activity in adipose tissue was not a spurious finding; it could be reproduced with an unrelated inhibitor of transcription, ␣-amanitin
Summary
Lipoprotein lipase (LPL) plays an important physiological role in regulating the release of fatty acids from triglyceriderich lipoproteins [1,2,3,4]. LPL is regulated at the level of gene expression in several physiological states: during fetal and early postnatal life, the enzyme is present in the liver but is suppressed [5]; in the mammary gland, the enzyme is switched on during lactation [6]; in macrophages, it is switched on when the cells are activated [7]; and in brown adipose tissue, the enzyme is switched on during cold adaptation [8]. LPL activity changes during the day according to the nutritional state. This appears to be mediated by posttranscriptional mechanisms. To delineate the regulatory mechanism, we determined the turnover rates for LPL mRNA, mass, and activity in rat adipose tissue in the fed and fasted states and explored whether the regulation during feeding, fasting, and refeeding requires synthesis of new mRNA or protein. The results point to a novel mechanism and a new perspective on the nutritional regulation of LPL in adipose tissue
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