Chronic ethanol feeding suppresses beta-adrenergic receptor-stimulated lipolysis in adipocytes isolated from epididymal fat

Abstract

Chronic ethanol consumption disrupts G protein-dependent signaling pathways in rat adipocytes. Because lipolysis in adipocytes is regulated by G protein-mediated cAMP signal transduction, we hypothesized that cAMP-regulated lipolysis may be vulnerable to long-term ethanol exposure. Male Wistar rats were fed a liquid diet containing ethanol as 35% of total calories or pair-fed a control diet that isocalorically substituted maltose dextrins for ethanol for 4 wk. Lipolysis was measured by glycerol release over 1 h with or without agonists in adipocytes isolated from epididymal fat. Chronic ethanol feeding decreased beta-adrenergic receptor-stimulated lipolysis, but had no effect on basal lipolysis. In response to beta-adrenergic activation, the early peak of cAMP accumulation was suppressed after ethanol feeding, although the basal cAMP concentration in adipocytes did not differ between pair- and ethanol-fed rats. The suppression in cAMP accumulation caused by ethanol feeding was associated with increased activity of phosphodiesterase 4. Chronic ethanol feeding also decreased beta-adrenergic receptor-stimulated protein kinase A activation and phosphorylation of its downstream proteins, perilipin A and hormone-sensitive lipase, the primary lipase-mediating lipolysis. In conclusion, these data suggest that chronic ethanol feeding increased phosphodiesterase 4 activity in adipocytes, resulting in decreased accumulation of cAMP in response to beta-adrenergic activation and a suppression of beta-adrenergic stimulation of lipolysis.

Fig. 1

Chronic ethanol feeding decreased β-adrenergic receptor-stimulated lipolysis in adipocytes isolated from epididymal fat. A, Adipocytes isolated from pair- and ethanol-fed rats were treated with or without increasing concentrations of isoproterenol. Basal lipolysis, measured as glycerol release over 1 h, did not differ in adipocytes from pair-fed (0.13 ± 0.02 μmol/10⁶ cells; n = 8) and ethanol-fed (0.14 ± 0.02 μmol/10⁶ cells; n = 8) rats. Values are the increase in glycerol release in response to isoproterenol over basal. Values represent means ± sem (n = 3) and were graphed using non-linear regression. B, Lipolysis in adipocytes was measured as described in Materials and Methods except that adenosine was included in the digestion and washing steps during adipocyte isolation. Adipocytes were treated with or without 1 μm isoproterenol in the presence of ADA (0.4 U/ml) and R-PIA (an A1 receptor agonist, 10 nm). Values are the increase in glycerol release in response to isoproterenol over release in the presence of ADA and R-PIA, and represent means ± sem (n = 4). *,P < 0.05, pair-fed vs. EtOH-fed. Iso, Isoproterenol; EtOH, ethanol.

Fig. 2

Chronic ethanol feeding suppressed β-adrenergic receptor-stimulated cAMP accumulation by increasing cAMP degradation via PDE4. Adipocytes isolated from pair- and ethanol-fed rats were pre-treated without (A) or with (B) 10 μm Ro20-1724 (RO) for 3 min, then 1 μm isoproterenol was added except the basal samples, and incubation was continued for another 0.5–15 min. Intracellular cAMP concentration was determined by cAMP enzyme immunoassay Biotrak system. Data are means ± sem (n = 3–7). *, P < 0.05 compared with 0 min within dietary group; +, P < 0.05 pair-fed vs. EtOH-fed. Iso, Isoproterenol; EtOH, ethanol.

Fig. 3

Chronic ethanol feeding increased basal activity of PDE4. Adipocytes isolated from pair- and ethanol-fed rats were incubated with or without 1 μm isoproterenol for 5 min. Cell homogenates were used to analyze PDE activity as described in Materials and Methods. Data represent means ± sem (n = 8 for basal samples or 4 for isoproterenol-treated samples). Values with different letters are significantly different (P < 0.05). EtOH, Ethanol.

Fig. 4

Chronic ethanol (EtOH) feeding did not increase the quantity of immunoreactive PDE4A, PDE4B, or PDE4D isoforms. Adipocytes isolated from pair- and ethanol-fed rats were homogenized in radio-immunoprecipitation buffer. After removal of the fat layer by centrifugation at 4000 × g for 3 min, the fluid fraction was prepared in SDS sample buffer and applied to a 6% SDS-polyacrylamide gel. PDE4 was immunoblotted using a PDE4 antibody (detecting all known PDE4 A and D variants) (A) and a PDE4B antibody (detecting all known PDE4B variants) (B). Rat testes were used as positive controls. A representative immunoblot from six experiments is shown. Asterisks indicate the immunoreactive PDE4 isoforms detected in rat testes, and the arrowheads indicate the corresponding bands detected in rat adipocytes.

Fig. 5

Chronic ethanol (EtOH) feeding decreased β-adrenergic receptor-stimulated PKA activity, but not maximal PKA activity. Adipocytes isolated from pair- and ethanol-fed rats were incubated with (A) or without (B) 1 μm isoproterenol (Iso) for 0–10 min. Cell homogenates were used to analyze PKA activity as described in Materials and Methods. Maximal PKA activity was assessed by the addition of 1.67 μm cAMP (B). Data represent means ± sem (n = 3– 8). *, P < 0.05 compared with own basal; +, P < 0.05, pair-fed vs. EtOH-fed.

Fig. 6

Phosphorylation of perilipin A and HSL were detected by a phospho-(Ser/Thr) PKA substrate antibody. Adipocytes isolated from pair-fed rats were treated with or without 1 μm isoproterenol for 5 or 10 min, and fat layer-associated protein samples or whole cell homogenates were prepared as indicated. A, Fat layer-associated protein samples were subjected to SDS-PAGE and immunoblotted with antibodies against perilipin A/B (Peri A/B) or phospho-(Ser/Thr) PKA substrate (p-Ser/Thr). B, Cell homogenates were subjected to SDS-PAGE and immunoblotted with phospho-(Ser/Thr) PKA substrate antibody.

Fig. 7

Chronic ethanol feeding reduced β-adrenergic receptor-stimulated phosphorylation of perilipin A and HSL. Adipocytes isolated from pair- and ethanol-fed rats were treated with or without 1 μm isoproterenol for 5 or 10 min, and fat layer-associated protein samples or whole cell homogenates were prepared as indicated. Phospho-perilipin A (p-Peri A; panel A) and phospho-HSL (p-HSL; panel B) were immunoblotted using a phospho-(Ser/Thr) PKA substrate antibody. A, A representative immunoblot of phospho-perilipin A is shown; the image shown for homogenate is from a longer exposure compared with the fat layer. With equal exposure times, phospho-perilipin A was 3.8 arbitrary units of density in fat layer compared with 1 in homogenate. B, A representative immunoblot for phospho-HSL is shown with equal exposure times for the fat layer and homogenate. Caveolin was used as a control for equal protein extraction from fat samples; ERK1 was used as a loading control in the cell homogenates. Values represent means ±sem (n = 4–6). *, P < 0.05, pair-fed vs. EtOH-fed. PF, Pair-fed; EF, ethanol-fed.

Fig. 8

Chronic ethanol feeding did not affect dibutyryl-cAMP-stimulated phosphorylation of perilipin A or HSL. Adipocytes isolated from pair- and ethanol-fed rats were treated with or without 0.5 mm dibutyryl-cAMP (db-cAMP) for 5 or 10 min, and fat layer-associated protein samples or whole cell homogenates were prepared as indicated. Phospho-perilipin A (p-Peri A; panel A) and phospho-HSL (pHSL; panel B) were immunoblotted using a phospho-(Ser/Thr) PKA substrate antibody. A representative immunoblot from four experiments is shown. PF, Pair-fed; EF, ethanol-fed.

Fig. 9

Chronic ethanol feeding impaired signaling downstream of phosphorylation of perilipin A and HSL. Adipocytes isolated from pair- and ethanol-fed rats were treated with or without increasing concentrations of dibutyryl-cAMP (db-cAMP; panel A) or pretreated with or without 10 μm Ro20-1724 (Ro) for 3 min before stimulation with 1 μm isoproterenol (Iso; panel B). Lipolysis was determined as glycerol release over 1 h. Values represent means ± sem (n = 3–8). *, P < 0.05, compared with own basal; +, P < 0.05, pair-fed vs. EtOH-fed. Values with different letters are significantly different (P < 0.05).