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. 2002 Dec 10;99(25):16309-13.
doi: 10.1073/pnas.222657499. Epub 2002 Nov 27.

Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation

Eva Tomas  1 , Tsu-Shuen Tsao, Asish K Saha, Heather E Murrey, Cheng cheng Zhang Cc, Samar I Itani, Harvey F Lodish, Neil B Ruderman
Affiliations

Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation

Eva Tomas et al. Proc Natl Acad Sci U S A. .

Abstract

gACRP30, the globular subunit of adipocyte complement-related protein of 30 kDa (ACRP30), improves insulin sensitivity and increases fatty acid oxidation. The mechanism by which gACRP30 exerts these effects is unknown. Here, we examined if gACRP30 activates AMP-activated protein kinase (AMPK), an enzyme that has been shown to increase muscle fatty acid oxidation and insulin sensitivity. Incubation of rat extensor digitorum longus (EDL), a predominantly fast twitch muscle, with gACRP30 (2.5 micro g/ml) for 30 min led to 2-fold increases in AMPK activity and phosphorylation of both AMPK on Thr-172 and acetyl CoA carboxylase (ACC) on Ser-79. Accordingly, concentration of malonyl CoA was diminished by 30%. In addition, gACRP30 caused a 1.5-fold increase in 2-deoxyglucose uptake. Similar changes in malonyl CoA and ACC were observed in soleus muscle incubated with gACRP30 (2.5 micro g/ml), although no significant changes in AMPK activity or 2-deoxyglucose uptake were detected. When EDL was incubated with full-length hexameric ACRP30 (10 micro g/ml), AMPK activity and ACC phosphorylation were not altered. Administration of gACRP30 (75 micro g) to C57 BL6J mice in vivo led to increased AMPK activity and ACC phosphorylation and decreased malonyl CoA concentration in gastrocnemius muscle within 15-30 min. Both in vivo and in vitro, activation of AMPK was the first effect of gACRP30 and was transient, whereas alterations in malonyl CoA and ACC occurred later and were more sustained. Thus, gACRP30 most likely exerts its actions on muscle fatty acid oxidation by inactivating ACC via activation of AMPK and perhaps other signal transduction proteins.

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Figures

Fig 1.
Fig 1.
Effect of incubation with gACRP30 on AMPK and related parameters. EDL muscles from 60-g rats were incubated for 30 min in the presence (+) or absence (−) of gACRP30 (2.5 μg/ml) and then frozen in liquid N2 until analyzed. (A) AMPK activity in immunoprecipitates obtained with antibody to the α2 isoform. (B) Western blots of AMPK phosphorylated at Thr-172 (active enzyme; p-AMPK), total AMPK abundance (anti-α1 and anti-α2 AMPK antibodies; AMPK), and ACC phosphorylated at Ser-79 (inhibited enzyme; p-ACC). (C) Concentration of malonyl CoA. Blots are representative of muscles of three to five animals. Measurements of AMPK activity and malonyl CoA concentration are means ± SE (n = 5). (D) Effect of gACRP30 on glucose transport in rat EDL. Muscles were incubated with (+) or without (−) gACRP30 (2.5 μg/ml) for 30 min. Glucose transport was then assessed in the absence of insulin based on the uptake of [3H]-2-deoxyglucose. Measurements of glucose transport are means ± SE (n = 6). Additional details are described in Materials and Methods. *, P < 0.05 vs. gACRP30 (−) group.
Fig 2.
Fig 2.
Time course of changes in AMPK activity and ACC phosphorylation in rat EDL incubated with and without gACRP30. AMPK activity is increased at 15 and 30 min and then returns to baseline values, whereas significant increases in ACC phosphorylation first become significant at 30 min and persist at 60 min. Results are means ± SE (n = 5) and are expressed as value for gACRP30 (−) group at each time point. *, P < 0.05 vs. 0 min.
Fig 3.
Fig 3.
Lack of effect of incubation with full-length ACRP30 on AMPK activity (A) and ACC phosphorylation (B). EDL were incubated for 30 min in the presence (+) or absence (−) of ACRP30 hexamer (10 μg/ml). Blots are representative of muscles of two to four animals. Measurements of AMPK activity are means ± SE (n = 4).
Fig 4.
Fig 4.
Effect of gACRP30 on AMPK activity and related parameters in the incubated rat soleus. Muscles were incubated for 30 min in the presence (+) and absence (−) of gACRP30 (2.5 μg/ml). (A) Malonyl CoA concentration. (B) Uptake of 2-deoxyglucose. (C) AMPK activity. (D) Phosphorylation of AMPK and ACC and AMPK abundance. Similar differences between the soleus and EDL (see Fig. 1) have been observed when these muscles are incubated with the AMPK activator AICAR (20). See the legend to Fig. 1 for additional details. Blots are representative of muscles of two to five animals. Measurements of AMPK activity and malonyl CoA concentration are means ± SE (n = 5). *, P < 0.05 vs. gACRP30 (−) group.
Fig 5.
Fig 5.
Effect of gACRP30 administered in vivo on AMPK activity and related parameters in mouse gastrocnemius muscle. Ten-week-old female C57BL/6J mice were fasted for 16 h, and then gACRP30 (75 μg per animal) (+) or saline [gACRP30 (−)] was injected into the retroorbital sinus. The rats were anesthetized with pentobarbital and muscle frozen in situ 15 or 30 min later. (A) AMPK activity (mean ± SE, n = 5) and malonyl CoA (n = 4). (B) Representative Western blots (n = 5) at 15 min. (C) Malonyl CoA concentration (mean ± SE, n = 5). (D) Representative Western blots of phosphorylated ACC at 30 min (n = 4). *, P < 0.05 vs. control.
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