A-769662 increased ACC phosphorylation in wild-type mouse embryonic fibroblasts, while the response was undetectable in AMPK-α1/α2 double-knockout cells. This supports AMPK dependence of the ACC endpoint under these conditions. [1]
AMPK activation does not necessarily suppress cell proliferation. In T98G, U87 and A172 glioma cells, 100 μM A769662 did not suppress proliferation through 72 h, despite increased AMPK signaling in the tested signaling models. [2]
Allosteric activation of native rat liver AMPK
Biochemical assay · Purified native rat liver AMPK: phosphorylated α1/α2 catalytic isoforms with β1 and γ1. [1]
A-769662 directly increased enzyme activity. Interaction with AMP was condition dependent: A-769662 added to saturating AMP gave a small further activation, whereas increasing AMP in saturating A-769662 reduced activity.
Literature-Reported Treatment Conditions
- Treatment concentration
- A-769662 titration; 1 μM in reciprocal AMP titration. EC50 below is an assay endpoint, not a cell treatment dose.
- Method
- Cell-free kinase activity assay
- Co-treatment
- Separate comparisons with or without 200 μM AMP; reciprocal AMP titration with or without 1 μM A-769662.
- Reported limits
- Assay duration and vehicle are not specified in the selected main-text assay description; no independent-replicate n assigned to fitted parameters.
Methods and source locations
Compound reported in the paper: A-769662; Compound label in the paper: A-769662
Evidence concerns the named research compound; it does not validate an Adooq lot, purity or formulation.
Figure 1A–B and caption; Results: A-769662 regulates heterotrimeric AMPK; Experimental procedures: AMPK preparations and assays
Half-maximal activation without added AMP: Activity versus A-769662 concentration fitted to activation model — Figure 1A caption
Maximal activation relative to fitted basal activity without added AMP: Concentration-response fit — Figure 1A caption
AMP titration in the presence of 1 μM A-769662: Reciprocal AMP concentration-response; comparison with activity at zero AMP — Figure 1B caption
Protection of AMPK Thr-172 phosphorylation
Biochemical assay · Purified rat liver AMPK incubated with recombinant protein phosphatase-2Cα. [1]
A-769662 protected AMPK Thr-172 phosphorylation against phosphatase-mediated loss; no-Mg2+ and AMP comparator conditions distinguish the assay response. This measures retention of AMPK phosphorylation, not direct phosphatase inhibition.
Literature-Reported Treatment Conditions
- Treatment concentration
- 1 μM A-769662
- Exposure duration
- Time course to 30 min (Figure 3B axis)
- Method
- Western blot for phospho-Thr-172, expressed relative to zero-time signal
- Co-treatment
- A-769662 condition contained phosphatase and 5 mM MgCl2; separate 200 μM AMP comparator and no-Mg2+ control.
- Reported limits
- Vehicle and replicate n are not given in the Figure 3B caption. No precise percentage protection inferred from graph.
Methods and source locations
Compound reported in the paper: A-769662; Compound label in the paper: A-769662
Evidence concerns the named research compound; it does not validate an Adooq lot, purity or formulation.
Figure 3B and caption; Results: Effects of AMP and A-769662 on dephosphorylation by protein phosphatase-2Cα
Retention of AMPK Thr-172 phosphorylation during phosphatase incubation: Immunoblot densitometry; normalized to zero time — Figure 3B, caption and associated Results
AMPK-dependent ACC phosphorylation in fibroblasts
Cell-based study · Immortalized wild-type and AMPK-α1−/−α2−/− mouse embryonic fibroblasts. [1]
A-769662 increased ACC phosphorylation in wild-type fibroblasts, with the response saturated at 100 μM. ACC phosphorylation was undetectable in double-knockout cells despite retained ACC expression. This establishes dependence of this endpoint on AMPK, not universal cellular selectivity.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0, 30, 100 and 300 μM A-769662
- Exposure duration
- 1 h
- Method
- Cell exposure followed by Western blotting and phospho-ACC densitometry
- Co-treatment
- Separate 2 mM phenformin comparator; not a combination treatment.
- Reported limits
- Cell-exposure vehicle is not specified in the relevant MEF methods/caption. Do not borrow 0.1% DMSO from the isolated-muscle protocol.
Methods and source locations
Compound reported in the paper: A-769662; Compound label in the paper: A-769662
Evidence concerns the named research compound; it does not validate an Adooq lot, purity or formulation.
Figure 5A–B and caption; Results: Phosphorylation of acetyl-CoA carboxylase in response to A-769662 requires AMPK; Cell culture and Western blotting methods
ACC phosphorylation in wild-type versus AMPK double-knockout fibroblasts: Phospho-ACC and total ACC immunoblots; means ± SEM of duplicates, representative of 2 or more experiments (caption) — Figure 5A–B and caption
AMPK signaling without suppression of glioma proliferation
Cell-based study · Human T98G, U87 and A172 glioma cells (proliferation); T98G and U87 (signaling time course). [2]
At 100 μM, A769662 increased AMPK/ACC phosphorylation but did not suppress glioma proliferation through 72 h. AICAR and metformin comparators suppressed proliferation at 48–72 h. These data limit generalization from AMPK activation to growth inhibition.
Literature-Reported Treatment Conditions
- Treatment concentration
- 100 μM A769662
- Exposure duration
- Signaling: 6, 24, 48 and 72 h; proliferation: 24, 48 and 72 h (Figure 1F–G)
- Method
- Drug added 24 h after seeding; immunoblot and viable-cell/proliferation analysis
- Co-treatment
- Separate 1 mM AICAR and 10 mM metformin comparator treatments; not coadministration.
- Reported limits
- Detailed cell medium, compound supplier/formulation and vehicle in SI were not retrieved. Figure 1G explicitly includes a control series; no vehicle composition is inferred. Main text describes Cell-titer-fluor and direct Trypan blue counting without assigning an exclusive assay to panel G.
Methods and source locations
Compound reported in the paper: A-769662; Compound label in the paper: A769662
Evidence concerns the named research compound; it does not validate an Adooq lot, purity or formulation.
Figure 1F–G and caption; Results: Glioma Cell Proliferation Is Suppressed by AICAR and Metformin but Not by the Direct AMPK Activator A769662; Experimental Procedures: Cell Culture and Growth Analysis
Glioma viable-cell accumulation versus control through 72 h: Parallel growth analysis; Figure 1 caption: representative of 3–6 independent experiments; error bars mean ± SD. Exact panel-specific n not given there. — Figure 1G, caption and associated Results
AMPK and ACC phosphorylation in treated glioma cells: Immunoblot time course; no numerical fold change inferred — Figure 1F and associated Results
