Abstract
The balance of lipid flux in adipocytes is controlled by the opposing actions of lipolysis and lipogenesis, which are controlled primarily by hormone-sensitive lipase and lipoprotein lipase (LPL), respectively. Catecholamines stimulate adipocyte lipolysis through reversible phosphorylation of hormone-sensitive lipase, and simultaneously inhibit LPL activity. However, LPL regulation is complex and previous studies have described translational regulation of LPL in response to catecholamines because of an RNA-binding protein that interacts with the 3'-untranslated region of LPL mRNA. In this study, we identified several protein components of an LPL RNA binding complex. Using an LPL RNA affinity column, we identified two of the RNA-binding proteins as the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), and A kinase anchoring protein (AKAP) 121/149, one of the PKA anchoring proteins, which has known RNA binding activity. To determine whether the C subunit was involved in LPL translation inhibition, the C subunit was depleted from the cytoplasmic extract of epinephrine-stimulated adipocytes by immunoprecipitation. This resulted in the loss of LPL translation inhibition activity of the extract, along with decreased RNA binding activity in a gel shift assay. To demonstrate the importance of the AKAPs, inhibition of PKA-AKAP binding with a peptide competitor (HT31) prevented epinephrine-mediated inhibition of LPL translation. C subunit kinase activity was necessary for LPL RNA binding and translation inhibition, suggesting that the phosphorylation of AKAP121/149 or other proteins was an important part of RNA binding complex formation. The hormonal activation of PKA results in the reversible phosphorylation of hormone-sensitive lipase, which is the primary mediator of adipocyte lipolysis. These studies demonstrate a dual role for PKA to simultaneously inhibit LPL-mediated lipogenesis through inhibition of LPL translation.
Highlights
The balance of lipid flux in adipocytes is controlled by the opposing actions of lipolysis and lipogenesis, which are controlled primarily by hormone-sensitive lipase and lipoprotein lipase (LPL), respectively
When adipocytes were treated with epinephrine, LPL translation was inhibited [12], and the epinephrine-treated cell extract caused a gel shift when added to a [32P]RNA fragment corresponding to LPL mRNA nucleotides 1512 to 1635 (Fig. 1A)
When the epinephrine-treated cell extract was applied to a DEAE column, as described under “Materials and Methods,” the RNA binding properties of the extract were predominantly found in the unbound fraction from the column. This material was dialyzed against a low salt buffer, and applied to a poly(U)Sepharose column containing the relevant binding region of the 3Ј-untranslated region (UTR) of the LPL mRNA
Summary
The balance of lipid flux in adipocytes is controlled by the opposing actions of lipolysis and lipogenesis, which are controlled primarily by hormone-sensitive lipase and lipoprotein lipase (LPL), respectively. Catecholamines stimulate adipocyte lipolysis through reversible phosphorylation of hormone-sensitive lipase, and simultaneously inhibit LPL activity. The hormonal activation of PKA results in the reversible phosphorylation of hormone-sensitive lipase, which is the primary mediator of adipocyte lipolysis. These studies demonstrate a dual role for PKA to simultaneously inhibit LPL-mediated lipogenesis through inhibition of LPL translation. Studies of 3T3 adipocytes demonstrated that the inhibition of LPL translation by epinephrine involved an RNA-binding protein that interacted with the proximal 3Ј-untranslated region (UTR) of the LPL mRNA [11]. The C subunit of PKA is likely part of an RNA binding complex, which involves A kinase anchoring protein (AKAP) 121/149, which is involved in binding the PKA holoenzyme, and which contains a known RNA binding domain [13]
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