Augmentation of lipolysis in adipocytes from fed rats, but not from starved rats, by inhibition of rolipram-sensitive phosphodiesterase 4

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Abstract

The sensitivity of adipocytes to lipolytic agents is increased after starvation. In this study, we found that LY294002, an inhibitor of phosphatidylinositol-3 kinase (PI3K), in the concentration of more than 50 μM potentiates lipolysis induced by adenosine deaminase in adipocytes from fed rats (f-adipocytes), but not from starved rats (s-adipocytes). It also enhanced the sensitivity to lipolytic action of isoproterenol in f-adipocytes much more than s-adipocytes. The target of LY294002 may be an anti-lipolytic regulator expressed in response to food intake. Since another PI3K inhibitor, wortmannin, or a phosphodiesterase 3 (PDE3) inhibitor, cilostamide, failed to cause any specific effect to f-adipocytes, the PI3K–PDE3B pathway cannot be a target of LY294002. We found that LY294002 inhibits efficiently the cytoplasmic PDE activity of adipocytes. Rolipram, a specific inhibitor of PDE4, also inhibited the cytoplasmic PDE and caused a preferential increase of lipolysis in f-adipocytes. LY294002 blunted the actions of rolipram on lipolysis and the PDE activity. LY294002 accelerated protein kinase A activation. These data suggest that the rolipram-sensitive PDE4 is an anti-lipolytic enzyme expressed according to food intake. LY294002 may potentiate lipolysis through inhibition of the PDE4.

Section snippets

Materials

The following inhibitors were used: LY294002, LY303511, wortmannin, rolipram, and cilostamide (Sigma). The PKA assay kit was purchased from CalbioChem and the kit, Eiken TG, was from Eiken Chemicals. Collagenase and snake venom (V7000) were purchased from Sigma and adenosine deaminase was from Boehringer–Mannheim.

Animals and adipocyte preparation

Male rats of the Charles River CD strain weighing 200–240 g (7-week-old) were used. Animals were fed a standard commercial diet ad libitum, or starved for 24 h. Both the fed and starved

Lipolysis

In this study, we found that adenosine deaminase causes lipolysis in a level of 4.4 ± 0.5 or 10.0 ± 1.4 μmol glycerol/ml packed cells/20 min for f-adipocytes or s-adipocytes, respectively (mean ± SE, n=4). Increase in lipolysis after starvation was apparent (p<0.02). Moreover, starvation enhanced the sensitivity to lipolytic action of isoproterenol, as shown by a decrease in the EC50 value determined by glycerol (Table 1). The EC50 assessed by free fatty acids also decreased, though not significant,

Discussion

The present study shows that rolipram causes a preferential increase of adenosine deaminase-induced lipolysis in f-adipocytes, indicating that the rolipram-sensitive PDE4 is an anti-lipolytic enzyme expressed according to food intake. There are two rolipram-binding sites in PDE4; a low-affinity rolipram-binding site, which exhibits IC50 values in the region of 0.1–1.0 μM, and a high-affinity rolipram-binding site, which exhibits IC50 values in the region of 1–50 nM [3]. A single site may change

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