Abstract
The post-transcriptional mechanisms by which feeding and insulin increase leptin production are poorly understood. Starvation of 6-7-week-old rats for 14 h decreased leptin mRNA level by only 22% but decreased plasma levels, adipose tissue leptin content, and release by over 75%. The decreased leptin with starvation was explained by >85% decrease in relative rates of leptin biosynthesis measured by metabolic labeling and immunoprecipitation. In vitro insulin treatment of adipose tissue from fed or starved rats for 2 h increased relative rates of leptin biosynthesis by 2-3-fold, and the effect was blocked by inhibition of phosphatidylinositol 3-kinase or mammalian target of rapamycin. Consistent with the hypothesis that feeding/insulin increases leptin translation, more leptin mRNA was associated with polysomes in adipose tissue of fed than starved rats, and in vitro incubation of adipose tissue of starved rats with insulin shifted leptin mRNA into polysomes. To assess the mechanisms regulating leptin translation, chimeric human leptin untranslated region (UTR) reporter constructs were transiently transfected into differentiated 3T3-L1 adipocytes. The 5'-UTR of leptin mRNA increased luciferase reporter activity 2-3-fold, whereas the full-length 3'-UTR (nucleotides 1-2804) was inhibitory (-65%). Sequences between nucleotides 462 and 1130 of the leptin 3'-UTR conferred most of the inhibitory effect. Insulin stimulated the expression of constructs that included both the full-length 5'-UTR and the inhibitory 3'-UTR, and the effect was blocked by inhibition of phosphatidylinositol 3-kinase or mammalian target of rapamycin. Our data suggest that insulin derepresses leptin translation by a mechanism that requires both the 5'-UTR and the 3'-UTR and may contribute to the increase in leptin production with feeding.
Highlights
Mi-Jeong Lee‡§, Rong-Ze Yang‡, Da-Wei Gong‡, and Susan K
In vitro insulin treatment of adipose tissue from fed or starved rats for 2 h increased relative rates of leptin biosynthesis by 2–3-fold, and the effect was blocked by inhibition of phosphatidylinositol 3-kinase or mammalian target of rapamycin
Overnight starvation decreased leptin mRNA levels in adipose tissue by 22%, yet decreased serum leptin by more than 85% (1.7 ng/ml versus not detectable Ͻ0.25 ng/ml, p Ͻ 0.01, n ϭ 4)
Summary
Animals—Male Wistar rats (5 weeks old) were purchased (Charles River Laboratory, Wilmington, MA) and housed with free access to laboratory chow and water until they reached the age of 6 –7 weeks. After 14 h of starvation or ad libitum feeding with water available, animals were anesthetized using CO2 and killed by decapitation, and trunk blood was collected
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