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

Abstract

Activation of AMP-activated protein kinase (AMPK) is considered a valid strategy for the treatment of type 2 diabetes. However, despite the importance of adipose tissue for whole-body energy homeostasis, the effect of AMPK activation in adipocytes has only been studied to a limited extent and mainly with the AMP-mimetic 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside (AICAR), which has limited specificity. The aim of this study was to evaluate the effect of the allosteric AMPK activators A‑769662 and 991 on glucose uptake in adipocytes. For this purpose, primary rat or human adipocytes, and cultured 3T3-L1 adipocytes, were treated with either of the allosteric activators, or AICAR, and basal and insulin-stimulated glucose uptake was assessed. Additionally, the effect of AMPK activators on insulin-stimulated phosphorylation of Akt and Akt substrate of 160 kDa was assessed. Furthermore, primary adipocytes from ADaM site binding drug-resistant AMPKb1 S108A knock-in mice were employed to investigate specificity of the drugs for the observed effects. Our results show that insulin-stimulated adipocyte glucose uptake was significantly reduced by A‑769662 but not 991, yet neither activator had any clear effects on basal or insulin-stimulated Akt/AS160 signaling. The use of AMPKb1 S108A mutant-expressing adipocytes revealed that the observed inhibition of glucose uptake by A‑769662 is most likely AMPK-independent, a finding which is supported by the rapid inhibitory effect A-769662 exerts on glucose uptake in 3T3-L1 adipocytes. These data suggest that AMPK activation per se does not inhibit glucose uptake in adipocytes and that the effects of AICAR and A-769662 are AMPK-independent.

The AMPK activator A-769662 inhibits human TASK3 potassium channels in an AMPK-independent manner

“…In HEK239 cells that stably over-expressed AMPK-b-1 S108A we observed a similar level of inhibition by high concentrations of A-769662 to that seen for cells expressing the wild-type. In short, A769662 inhibits hTASK-3 channels in an off-target, AMPK-independent manner, adding to previous reports of off-target inhibition of the sodium-potassium ATPase (36) and glucose uptake (37). Contrary to this, A-769662 has been shown to be without effect on TASK-1/3 currents in rat carotid body type I cells (21,22) and therefore may exhibit selectivity for hTASK-3 over rat TASK-3 species dependency (human vs rat).…”

Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

“…After three cell lines were treated with a selective AMPK activator (A-769662) or an AMPK inhibitor (compound C), we found that both the inhibition and activation of AMPKα resulted in decreased levels of lamin A/C and pLamin A/C-S22 ( Figure 5 C). Recent reports have revealed that A-769662 inhibits adipocyte differentiation, proteasomal activity, cell growth and DNA replication via an AMPK-independent mechanism [ 42 ]. As an important molecule for differentiation, lamin A/C is presumably a direct target of A-769662.…”

AMP-activated protein kinase activation suppresses leptin expression independently of adipogenesis in primary murine adipocytes

“…These results may vary due to the primary nature of our cell model, or may be due to the off-target effects of AICAR. Indeed, the effect of both AICAR and A-769662, a small molecule AMPK activator, have been shown to modulate glucose uptake in adipocytes in an AMPK-independent manner [ 55 ], suggesting the effects of AICAR on TGFβ/Wnt may also be independent of AMPK. Further studies using AMPK knockout models are required to evaluate these differences, and to investigate the role of AMPK in TGFβ/Wnt signalling in adipocytes under inflammatory conditions, where TGFβ signalling is elevated.…”