| 1. |
- Ahwazi, Danial, et al.
(författare)
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Investigation of the specificity and mechanism of action of the ULK1/AMPK inhibitor SBI-0206965
- 2021
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Ingår i: Biochemical Journal. - 0264-6021. ; 478:15, s. 2977-2997
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Tidskriftsartikel (refereegranskat)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. |
- Göransson, Olga, et al.
(författare)
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Metabolic control by AMPK in white adipose tissue
- 2023
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Ingår i: Trends in Endocrinology and Metabolism. - 1043-2760. ; 34:11, s. 704-717
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Forskningsöversikt (refereegranskat)abstract
- White adipose tissue (WAT) plays an important role in the integration of whole-body metabolism by storing fat and mobilizing triacylglycerol when needed. The released free fatty acids can then be oxidized by other tissues to provide ATP. AMP-activated protein kinase (AMPK) is a key regulator of metabolic pathways, and can be targeted by a new generation of direct, small-molecule activators. AMPK activation in WAT inhibits insulin-stimulated lipogenesis and in some situations also inhibits insulin-stimulated glucose uptake, but AMPK-induced inhibition of β-adrenergic agonist-stimulated lipolysis might need to be re-evaluated in vivo. The lack of dramatic effects of AMPK activation on basal metabolism in WAT could be advantageous when treating type 2 diabetes with pharmacological pan-AMPK activators.
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| 3. |
- Kopietz, Franziska, et al.
(författare)
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A-769662 inhibits adipocyte glucose uptake in an AMPK-independent manner
- 2021
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Ingår i: The Biochemical journal. - 0264-6021. ; 478:3, s. 633-646
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Tidskriftsartikel (refereegranskat)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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| 4. |
- Kopietz, Franziska
(författare)
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AMP-activated protein kinase in adipose tissue
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 5. |
- Kopietz, Franziska, et al.
(författare)
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AMPK activation by A-769662 and 991 does not affect catecholamine-induced lipolysis in human adipocytes
- 2018
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Ingår i: American Journal of Physiology - Endocrinology and Metabolism. - : American Physiological Society. - 1522-1555 .- 0193-1849. ; 315:5, s. 1075-1085
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Tidskriftsartikel (refereegranskat)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-D-ribofuranoside (AICAR), suggest an anti-lipolytic role of AMPK in adipocytes. The aim of this work was to re-investigate 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, was then monitored. Our results show that both A-769662 and 991 activated AMPK to a level which was similar to, or greater than that induced by AICAR. In contrast to AICAR, which as expected was anti-lipolytic, neither A-769662 nor 991 affected lipolysis in human adipocytes, although 991 treatment lead 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 anti-lipolytic effect observed with AICAR in previous studies is, at least to some extent, AMPK-independent.
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| 6. |
- Kopietz, Franziska, et al.
(författare)
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AMPKβ isoform expression patterns in various adipocyte models and in relation to body mass index
- 2022
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Ingår i: Frontiers in Physiology. - : Frontiers Media SA. - 1664-042X. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- AMP-activated protein kinase (AMPK) activation is considered a useful strategy for the treatment of type 2 diabetes (T2D). It is unclear whether the expression and/or activity of AMPK in adipocytes is dysregulated in obesity. Also, the expression/activity pattern of AMPKβ isoforms, which are targets for AMPK activators, in adipocytes remains elusive. In this study we show that the two AMPKβ isoforms make roughly equal contributions to AMPK activity in primary human and mouse adipocytes, whereas in cultured 3T3-L1 adipocytes of mouse origin and in primary rat adipocytes, β1-associated activity clearly dominates. Additionally, we found that obesity is not associated with changes in AMPK subunit expression or kinase activity in adipocytes isolated from subcutaneous adipose tissue from individuals with various BMI.
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| 7. |
- Kopietz, Franziska, et al.
(författare)
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Inhibition of AMPK activity in response to insulin in adipocytes : involvement of AMPK pS485, PDEs, and cellular energy levels
- 2020
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Ingår i: American Journal of Physiology - Endocrinology and Metabolism. - : American Physiological Society. - 1522-1555 .- 0193-1849. ; 319:3, s. 459-471
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Tidskriftsartikel (refereegranskat)abstract
- Insulin resistance in obesity and type 2 diabetes has been shown to be associated with decreased de novo fatty acid (FA) synthesis in adipose tissue. It is known that insulin can acutely stimulate FA synthesis in adipocytes; however, the mechanisms underlying this effect are unclear. The rate-limiting step in FA synthesis is catalyzed by acetyl-CoA carboxylase (ACC), known to be regulated through inhibitory phosphorylation at S79 by the AMP-activated protein kinase (AMPK). Previous results from our laboratory showed an inhibition of AMPK activity by insulin, which was accompanied by PKB-dependent phosphorylation of AMPK at S485. However, whether the S485 phosphorylation is required for insulin-induced inhibition of AMPK or other mechanisms underlie the reduced kinase activity is not known. To investigate this, primary rat adipocytes were transduced with a recombinant adenovirus encoding AMPK-WT or a nonphosphorylatable AMPK S485A mutant. AMPK activity measurements by Western blot analysis and in vitro kinase assay revealed that WT and S485A AMPK were inhibited to a similar degree by insulin, indicating that AMPK S485 phosphorylation is not required for insulin-induced AMPK inhibition. Further analysis suggested an involvement of decreased AMP-to-ATP ratios in the insulin-induced inhibition of AMPK activity, whereas a possible contribution of phosphodiesterases was excluded. Furthermore, we show that insulin-induced AMPK S485 phosphorylation also occurs in human adipocytes, suggesting it to be of an importance yet to be revealed. Altogether, this study increases our understanding of how insulin regulates AMPK activity, and with that, FA synthesis, in adipose tissue.
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| 8. |
- Neuhaus, Mathis, et al.
(författare)
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EHD2 regulates plasma membrane integrity and downstream insulin receptor signalling events
- 2023
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Ingår i: Molecular Biology of the Cell. - 1939-4586. ; 34:12
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Tidskriftsartikel (refereegranskat)abstract
- Adipocyte dysfunction is a crucial driver of insulin resistance and type 2 diabetes. We identified EH domain-containing protein 2 (EHD2) as one of the most highly upregulated genes at the early stage of adipose tissue expansion. EHD2 is a dynamin-related ATPase influencing several cellular processes, including membrane recycling, caveolae dynamics and lipid metabolism. Here, we investigated the role of EHD2 in adipocyte insulin signalling and glucose transport. Using C57BL6/N EHD2 knockout mice under short-term high-fat diet conditions and 3T3-L1 adipocytes we demonstrate that EHD2 deficiency is associated with deterioration of insulin signal transduction and impaired insulin-stimulated GLUT4 translocation. Furthermore, we show that lack of EHD2 is linked with altered plasma membrane lipid and protein composition, reduced insulin receptor expression, and diminished insulin-dependent SNARE protein complex formation. In conclusion, these data highlight the importance of EHD2 for the integrity of the plasma membrane milieu, insulin receptor stability, and downstream insulin receptor signalling events, involved in glucose uptake and ultimately underscore its role in insulin resistance and obesity.
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| 9. |
- Pålbrink, Ann-Ki, et al.
(författare)
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Inner ear is a target for insulin signaling and insulin resistance : evidence from mice and auditory HEI-OC1 cells
- 2020
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Ingår i: BMJ open diabetes research & care. - : BMJ. - 2052-4897. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: The mechanisms underlying the association between diabetes and inner ear dysfunction are not known yet. The aim of the present study is to evaluate the impact of obesity/insulin resistance on inner ear fluid homeostasis in vivo, and to investigate whether the organ of Corti could be a target tissue for insulin signaling using auditory House Ear Institute-Organ of Corti 1 (HEI-OC1) cells as an in vitro model.METHODS: High fat diet (HFD) fed C57BL/6J mice were used as a model to study the impact of insulin resistance on the inner ear. In one study, 12 C57BL/6J mice were fed either control diet or HFD and the size of the inner ear endolymphatic fluid compartment (EFC) was measured after 30 days using MRI and gadolinium contrast as a read-out. In another study, the size of the inner ear EFC was evaluated in eight C57BL/6J mice both before and after HFD feeding, with the same techniques. HEI-OC1 auditory cells were used as a model to investigate insulin signaling in organ of Corti cells.RESULTS: HFD feeding induced an expansion of the EFC in C57BL/6J mice, a hallmark of inner ear dysfunction. Insulin also induced phosphorylation of protein kinase B (PKB/Akt) at Ser473, in a PI3-kinase-dependent manner. The phosphorylation of PKB was inhibited by isoproterenol and IBMX, a general phosphodiesterase (PDE) inhibitor. PDE1B, PDE4D and the insulin-sensitive PDE3B were found expressed and catalytically active in HEI-OC1 cells. Insulin decreased and AICAR, an activator of AMP-activated protein kinase, increased the phosphorylation at the inhibitory Ser79 of acetyl-CoA carboxylase, the rate-limiting enzyme in de novo lipogenesis. Furthermore, the activity of hormone-sensitive lipase, the rate-limiting enzyme in lipolysis, was detected in HEI-OC1 cells.CONCLUSIONS: The organ of Corti could be a target tissue for insulin action, and inner ear insulin resistance might contribute to the association between diabetes and inner ear dysfunction.
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| 10. |
- Säll, Johanna, et al.
(författare)
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Insulin induces Thr484 phosphorylation and stabilization of SIK2 in adipocytes
- 2019
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Ingår i: Cellular Signalling. - : Elsevier BV. - 1873-3913 .- 0898-6568. ; 55, s. 73-80
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Tidskriftsartikel (refereegranskat)abstract
- AIMS/HYPOTHESIS: Salt-inducible kinase 2 (SIK2) is downregulated in adipose tissue from obese or insulin-resistant individuals and inhibition of SIK isoforms results in reduced glucose uptake and insulin signalling in adipocytes. However, the regulation of SIK2 itself in response to insulin in adipocytes has not been studied in detail. The aim of our work was to investigate effects of insulin on various aspects of SIK2 function in adipocytes.METHODS: Primary adipocytes were isolated from human subcutaneous and rat epididymal adipose tissue. Insulin-induced phosphorylation of SIK2 and HDAC4 was analyzed using phosphospecific antibodies and changes in the catalytic activity of SIK2 with in vitro kinase assay. SIK2 protein levels were analyzed in primary adipocytes treated with the proteasome inhibitor MG132.RESULTS: We have identified a novel regulatory pathway of SIK2 in adipocytes, which involves insulin-induced phosphorylation at Thr484. This phosphorylation is impaired in individuals with a reduced insulin action. Insulin stimulation does not affect SIK2 catalytic activity or cellular activity towards HDAC4, but is associated with increased SIK2 protein levels in adipocytes.CONCLUSION/INTERPRETATION: Our data suggest that downregulation of SIK2 in the adipose tissue of insulin-resistant individuals can partially be caused by impaired insulin signalling, which might result in defects in SIK2 expression and function.
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