Abstract
AMP-activated protein kinase (AMPK) is a heterotrimer of α-catalytic and β- and γ-regulatory subunits that acts to regulate cellular and whole-body nutrient metabolism. The key role of AMPK in sensing energy status has led to significant interest in AMPK as a therapeutic target for dysfunctional metabolism in type 2 diabetes, insulin resistance and obesity. Despite the actions of AMPK in the liver and skeletal muscle being extensively studied, the role of AMPK in adipose tissue and adipocytes remains less well characterised. Small molecules that selectively influence AMPK heterotrimers containing specific AMPKβ subunit isoforms have been developed, including MT47-100, which selectively inhibits complexes containing AMPKβ2. AMPKβ1 and AMPKβ2 are the principal AMPKβ subunit isoforms in rodent liver and skeletal muscle, respectively, yet the contribution of specific AMPKβ isoforms to adipose tissue function, however, remains largely unknown. This study therefore sought to determine the contribution of AMPKβ subunit isoforms to adipocyte biology, focussing on adipogenesis. AMPKβ2 was the principal AMPKβ isoform in 3T3-L1 adipocytes, isolated rodent adipocytes and human subcutaneous adipose tissue, as assessed by the contribution to total cellular AMPK activity. Down-regulation of AMPKβ2 with siRNA inhibited lipid accumulation, cellular adiponectin levels and adiponectin secretion during 3T3-L1 adipogenesis, whereas down-regulation of AMPKβ1 had no effect. Incubation of 3T3-L1 cells with MT47-100 selectively inhibited AMPK complexes containing AMPKβ2 whilst simultaneously inhibiting cellular lipid accumulation as well as cellular levels and secretion of adiponectin. Taken together, these data indicate that increased expression of AMPKβ2 is an important feature of efficient adipogenesis.
Highlights
AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that responds to increases in AMP relative to ATP, resulting from such stimuli as hypoxia, nutrient deprivation and ischaemia
To examine whether levels of AMPK isoforms were altered during adipogenesis of 3T3-L1 adipocytes, levels of each AMPK subunit isoform were assessed by immunoblotting in 3T3-L1 preadipocytes and 12 days postdifferentiation into adipocytes
There was a marked 2-fold increase in the levels of AMPKβ2 relative to AMPKβ1 as 3T3-L1 preadipocytes were differentiated into adipocytes, observed by 4 days post-differentiation assessed with AMPKβ isoformspecific antibodies (Figure 1a,c,e,f )
Summary
AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that responds to increases in AMP (or ADP) relative to ATP, resulting from such stimuli as hypoxia, nutrient deprivation and ischaemia. The β-subunit contains a C-terminal domain that forms the core of the heterotrimeric complex, in addition to a carbohydrate-binding domain of poorly defined function [1,2]. A number of direct AMPK activators, such as A769662 and compound 991, have been developed, which have been demonstrated to bind a site between the β-subunit carbohydrate-binding domain and the α-subunit [7]. Compounds binding at this site show marked selectivity for AMPK complexes containing β1 rather than β2 [8], such that pharmacological targeting of specific tissues based on their AMPKβ isoform expression is feasible
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