Abstract
Adipose tissue is a major site of glycerol production in response to energy balance. However, molecular basis of glycerol release from adipocytes has not yet been elucidated. We recently cloned a novel member of the aquaporin family, aquaporin adipose (AQPap), which has glycerol permeability. The current study was designed to examine the hypothesis that AQPap serves as a glycerol channel in adipocytes. Adipose tissue expressed AQPap mRNA in high abundance, but not the mRNAs for the other aquaglyceroporins, AQP3 and AQP9, indicating that AQPap is the only known aquaglyceroporin expressed in adipose tissue. Glycerol release from 3T3-L1 cells was increased during differentiation in parallel with AQPap mRNA levels and suppressed by mercury ion, which inhibits the function of AQPs, supporting AQPap functions as a glycerol channel in adipocytes. Fasting increased and refeeding suppressed adipose AQPap mRNA levels in accordance with plasma glycerol levels and oppositely to plasma insulin levels in mice. Insulin dose-dependently suppressed AQPap mRNA expression in 3T3-L1 cells. AQPap mRNA levels and adipose glycerol concentrations measured by the microdialysis technique were increased in obese mice with insulin resistance. Accordingly, negative regulation of AQPap expression by insulin was impaired in the insulin-resistant state. Exposure of epinephrine translocated AQPap protein from perinuclear cytoplasm to the plasma membrane in 3T3-L1 adipocytes. These results strongly suggest that AQPap plays an important role in glycerol release from adipocytes.
Highlights
Adipose tissue is a major site of glycerol production in response to energy balance
Glycerol release from 3T3-L1 cells was increased during differentiation in parallel with aquaporin adipose (AQPap) mRNA levels and suppressed by mercury ion, which inhibits the function of AQPs, supporting AQPap functions as a glycerol channel in adipocytes
We examined the expression of mRNAs for AQP3 and AQP9, which are other members of aquaglyceroporins shown to have the ability to transport glycerol
Summary
Animals and Cells—Eight-week-old male C57BL/6J, C57BL/KsJ (dbϩ/ϩm), and C57BL/KsJ (dbϩ/dbϩ) mice were purchased from Clea Japan, Inc. Effect of fasting and feeding on AQPap mRNA expression in adipose tissue was examined in male C57BL/KsJ (dbϩ/ϩm) mice (n ϭ 8, each). 3T3-L1 cells on day 9 after differentiation were washed twice with PBS followed by incubation with PBS containing various concentration of HgCl2 for 5 min. Total RNAs from various tissues of overnight fasted C57BL/6J mice were subjected to Northern blot analysis (10 g/lane). Crude membrane fractions of 3T3-L1 cells and mouse adipose tissues were prepared as follows. Cells were washed with PBS and incubated with biotinylated swine serum to anti-rabbit immunogloblins in PBS containing 5% BSA for 30 min at room temperature. The significance of the difference between the mean values of the groups was evaluated by Student’s t test or analysis of variance (ANOVA) with Fisher’s PLSD test
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