| 1. |
- Ahwazi, Danial, et al.
(author)
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Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965
- 2021
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In: Biochemical Journal. - 0264-6021. ; 478:15, s. 2977-2997
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Journal article (peer-reviewed)abstract
- SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective AMP-activated protein kinase (AMPK) inhibitor relative to the widely used, but promiscuous inhibitor Compound C/ Dorsomorphin. Here, we studied the effects of SBI-0206965 on AMPK signalling and metabolic readouts in multiple cell types, including hepatocytes, skeletal muscle cells and adipocytes. We observed SBI-0206965 dose dependently attenuated AMPK activator (991)-stimulated ACC phosphorylation and inhibition of lipogenesis in hepatocytes. SBI-0206965 (≥25 μM) modestly inhibited AMPK signalling in C2C12 myotubes, but also inhibited insulin signalling, insulin-mediated/AMPK-independent glucose uptake, and AICA-riboside uptake. We performed an extended screen of SBI-0206965 against a panel of 140 human protein kinases in vitro, which showed SBI-0206965 inhibits several kinases, including members of AMPK-related kinases (NUAK1, MARK3/4), equally or more potently than AMPK or ULK1. This screen, together with molecular modelling, revealed that most SBI-0206965-sensitive kinases contain a large gatekeeper residue with a preference for methionine at this position. We observed that mutation of the gatekeeper methionine to a smaller side chain amino acid (threonine) rendered AMPK and ULK1 resistant to SBI-0206965 inhibition. These results demonstrate that although SBI-0206965 has utility for delineating AMPK or ULK1 signalling and cellular functions, the compound potently inhibits several other kinases and critical cellular functions such as glucose and nucleoside uptake. Our study demonstrates a role for the gatekeeper residue as a determinant of the inhibitor sensitivity and inhibitor-resistant mutant forms could be exploited as potential controls to probe specific cellular effects of SBI-0206965.
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| 2. |
- Kopietz, Franziska, et al.
(author)
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A-769662 inhibits adipocyte glucose uptake in an AMPK-independent manner
- 2021
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In: The Biochemical journal. - 0264-6021. ; 478:3, s. 633-646
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Journal article (peer-reviewed)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.
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| 3. |
- Kopietz, Franziska
(author)
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AMP-activated protein kinase in adipose tissue
- 2021
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Doctoral thesis (other academic/artistic)abstract
- The AMP-activated protein kinase (AMPK) is a ubiquitously expressed kinase, which is activated in response to depletion of cellularenergy levels. Once active, it functions to alter cellular metabolism in a way that leads to restoration of energy levels. Therefore,AMPK is described as the key regulator of cellular energy homeostasis. Over the past years, AMPK activation has gained increasingattention as a promising strategy for the treatment of type 2 diabetes (T2D). However, this view is mainly based on findings obtainedin muscle and liver tissue. Although muscle and liver are important players in the regulation of whole-body glucose homeostasis andare therefore valid targets in the treatment of T2D, there is also evidence that dysregulation of adipose tissue function during obesityis a key factor in the development of insulin resistance and T2D. Despite that, AMPK is only studied to a limited extent in adiposetissue. Therefore, the aim of this thesis was to contribute to an increased understanding not only of the effect of AMPK activationon adipocyte metabolism, but also the regulation and expression of AMPK in adipocytes.The first part of this work (Paper I + II) focuses on potential effects of AMPK activation on adipocyte metabolism. By employingAMPK activators of a new generation, A-769662 and 991, as well as a mutant mouse model, we provide evidence that AMPKactivation neither affects lipolysis nor glucose uptake in human adipocytes. Thereby, our results suggest that the previously observedeffects are likely to be AMPK-independent and challenge the up to now prevalent view of an anti-lipolytic and glucose uptakeinhibitingeffect of AMPK activation in adipocytes.The second part (Paper III) constitutes a quantitative analysis of the expression and contribution to overall AMPK kinase activity ofthe two regulatory AMPKβ-subunit isoforms. Moreover, we have performed a correlation analysis to investigate potential alterationsin AMPK activity and expression in relation to BMI. Our results suggest that AMPKb1 is the main isoform expressed in humanadipocytes. However, it seems like AMPK activity/and expression does not correlate with human obesity/BMI.In the last part of the work presented here (Paper IV), we have assessed potential mechanisms underlying the inhibitory effect ofinsulin on AMPK activity. While we refute the previously suggested involvement of AMPK Ser485 phosphorylation, we revealed anoverall decrease in cellular energy levels in response to adipocyte insulin stimulation which might mediate the observed inhibition ofAMPK activity.
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