AMPK activation by A-769662 and 991 does not affect catecholamine-induced lipolysis in human adipocytes

Abstract

Activation of AMP-activated protein kinase (AMPK) is considered an attractive strategy for the treatment of type 2 diabetes. Favorable metabolic effects of AMPK activation are mainly observed in skeletal muscle and liver tissue, whereas the effects in human adipose tissue are only poorly understood. Previous studies, which largely employed the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), suggest an antilipolytic role of AMPK in adipocytes. The aim of this work was to reinvestigate the role of AMPK in the regulation of lipolysis, using the novel allosteric small-molecule AMPK activators A-769662 and 991, with a focus on human adipocytes. For this purpose, human primary subcutaneous adipocytes were treated with A-769662, 991, or AICAR, as a control, before being stimulated with isoproterenol. AMPK activity status, glycerol release, and the phosphorylation of hormone-sensitive lipase (HSL), a key regulator of lipolysis, were then monitored. Our results show that both A-769662 and 991 activated AMPK to a level that was similar to, or greater than, that induced by AICAR. In contrast to AICAR, which as expected was antilipolytic, neither A-769662 nor 991 affected lipolysis in human adipocytes, although 991 treatment led to altered HSL phosphorylation. Furthermore, we suggest that HSL Ser660 is an important regulator of lipolytic activity in human adipocytes. These data suggest that the antilipolytic effect observed with AICAR in previous studies is, at least to some extent, AMPK independent.

AMP-activated protein kinase complexes containing the β2 regulatory subunit are up-regulated during and contribute to adipogenesis

“…The demonstration that AMPKα1 is the principal catalytic subunit isoform in terms of total cellular AMPK activity in 3T3-L1 adipocytes, isolated rodent adipocytes and human SCAT agrees with previous reports [38–41]. Furthermore, AMPK complexes containing β2 account for the majority of total cellular AMPK activity in mouse and rat adipocytes isolated from two separate WAT depots as well as human SCAT, phenocopying 3T3-L1 adipocytes and suggesting the switch from complexes containing AMPKβ1 to those containing AMPKβ2 during adipogenesis in 3T3-L1 cells reflects normal physiology.…”

“…This suggests either that the antibody that recognises both AMPKβ isoforms does not exhibit equal affinity for each isoform or that the specific activity of complexes containing AMPKβ2 is far greater than those containing AMPKβ1. During the preparation of this manuscript, it was reported that the levels of AMPKβ2 relative to AMPKβ1 do not change between 3T3-L1 preadipocytes and 3T3-L1 adipocytes using the same antibody, although mouse and human adipocytes exhibited substantial levels of AMPKβ2 immunoreactivity [41]. The reasons for this discrepancy are uncertain.…”

A-769662 inhibits adipocyte glucose uptake in an AMPK-independent manner

“…1B), which was expected as 991 is more potent and binds AMPK 5-10-fold tighter than A-769662 in cell-free assays (12). These results are in line with our previous study showing efficient activation of AMPK by A-769662 and 991 in adipocytes from different primary adipocyte models (18). In addition to rat adipocytes, the effect of AMPK activation on glucose uptake was also investigated in human adipocytes isolated from subcutaneous adipose tissue.…”

“…However, despite the role of adipose tissue in the development of insulin resistancea key feature of T2D, the regulation of adipocyte metabolism by AMPK has been poorly characterized (16,17). While we recently described the effect of AICAR and the ADaM site AMPK activators on lipolysis, adipocyte glucose uptake has only been studied using AICAR (18)(19)(20). To date, the results obtained with AICAR suggest increased basal and decreased insulin-stimulated glucose uptake in cultured 3T3-L1 adipocytes (20,21).…”

The Future of Metformin in the Prevention of Diabetes-Related Osteoporosis

“…Another pathway that has an effect of AMPK is calmodulin-dependent protein kinase kinase (CaMKKbeta), which activates via similar phosphorylation activity [ 33 - 34 ]. A more recent finding suggests some molecules can directly activate AMPK such as a small molecule numbered as A-769662 and an enzyme called 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) [ 35 ]. Also, fructose-1, 6-bisphosphate can have a positive mediation effect on AMPK, which can lower liver glucose production [ 36 ].…”