[
MicroPubl Biol,
2022]
AMP-activated protein kinase (AMPK) is a key metabolic regulator which responds to changes in the AMP:ATP ratio within cells. In response to high AMP levels, AMPK promotes a metabolic shift towards increased catabolism and autophagy to restore cellular energy and maintain homeostasis. In C. elegans, AMPK is important for controlling a multitude of functions including metabolism, reproductive health, and lifespan. AMPK is a heterotrimeric protein consisting of α catalytic, β linker, and γ regulatory subunits. Active AMPK is characterised by phosphorylation of the α subunit. It is also regulated allosterically by the nucleotide AMP binding within the γ subunit. C. elegans have five different AMPKγ subunits and their primary amino acid sequence implies two different modes of AMP-binding. Modifying the ability of AMPKγ to bind adenine nucleotides could directly impact how effectively AMPK manages energy homeostasis. Despite the importance of the γ subunit, most C. elegans AMPK research has focused on the catalytic α subunit. Here, we genetically dissect the functional role of the different γ subunits in relation to physiology and lifespan. We show that in normal animals, three of these γ subunits (
aakg-1,
aakg-2, and
aakg-3 ) are required for normal responses to AMP, and contribute to normal fecundity and lifespan.
[
Mol Biol Cell,
2006]
Monitoring Editor: Jennifer Lippincott-Schwartz Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, since fusion can proceed in
spe-9 fertilization mutants but is blocked by RNAi-mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1-dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.
Sedore, Christine A., Johnson, Erik, Banse, Stephen A., Lithgow, Gordon J., Driscoll, Monica, Coleman-Hulbert, Anna L., Guo, Max, Phillips, Patrick C.
[
microPublication Biology,
2020]
The Caenorhabditis Intervention Testing Program (CITP) is a multi-institutional, National Institutes of Aging (NIA)-funded consortium charged with identifying chemical compounds that robustly extend lifespan in a genetically diverse panel of Caenorhabditis strains. Compounds are prioritized for screening if they are highly ranked via computational prediction for lifespan or healthspan effects (Coleman-Hulbert et al. 2019), if they are predicted to engage known lifespan regulating pathways, or if they have previously been reported as extending lifespan or healthspan in model systems (Lucanic et al. 2017). -guanidinopropionic acid (-GPA) is a creatine analog (Shields and Whitehair 1973), commonly used as a dietary supplement, and has been shown to extend lifespan in Drosophila under stress via 5′ AMP-activated protein kinase (AMPK) activity (Yang et al. 2015). The AMPK pathway is conserved in nematodes and humans (Apfeld et al. 2004) and is involved in multiple pathways affecting stress response and metabolism (Wang et al. 2012).
We assayed lifespan in response to -GPA exposure in three Caenorhabditis species using our previously published workflow (Lucanic et al. 2017). In brief, worms were age-synchronized by timed egg-lays on standard 60 mm diameter Nematode Growth Media (NGM) plates and transferred at a density of 50 individuals per 35 mm treated plate in triplicate when they reached adulthood (for control plates, there were six replicates of 50 animals each). -GPA (Sigma-Aldrich) was dissolved in water and diluted appropriately such that addition of 125 l of solution to 35 mm diameter plates containing NGM with lawns of E. coli OP50-1 and 51 m FUdR would generate the following final -GPA concentrations: 0.1 M, 1 M, 10 M, 50 M, 100 M, 500 M, 1 mM, 10 mM and 300 mM. Worms were maintained at 20 C and moved to fresh plates on the first, second, and fourth (C. tropicalis) or fifth (C. elegans and C. briggsae) day of adulthood, then once weekly afterward. Due to our previous experience with compound interventions that potentially alter bacterial viability through transient pH changes upon plate treatment (Banse et al. 2019), we investigated the effects of -GPA on our assay plates. At the above listed concentrations, -GPA-treated plates had a pH of 6.5 and the bacterial lawns survived treatment when tested by replica plating. Thrice weekly, we observed animals for spontaneous movement or movement after gentle perturbation with a 0.2 mm diameter platinum wire. Death was scored as a lack of movement.
Our results indicate that -GPA does not extend lifespan in the three nematode species at the concentrations tested here; in fact, in only one instance was an effect detected and, in that case, the compound reduced lifespan (Fig. 1). This conclusion is based on one biological replicate per concentration and, as such, could be considered preliminary. While interventions may be ineffective due to a range of causes, including permeability barriers, compound stability in vivo, and metabolism by the bacterial food source, we believe that the lack of response in this study was due to a lack of physiological relevance in C. elegans. In Drosophila and mammalian models, -GPA reduces the level of intracellular phosphocreatine that can be used by creatine kinases to regenerate ATP (Oudman et al. 2013; Yang et al. 2015) resulting in a decreased cellular ATP/AMP ratio, which activates AMPK and ultimately increases lifespan and stress-resistance. Our a priori expectation for lifespan extension in C. elegans was built on the observations that: (1) creatine is reported as detectible in C. elegans (Atherton et al. 2008; Jones et al. 2012; Wan et al. 2017); (2) C. elegans has a creatine-like kinase, ARGK-1, whose activity modulates AMPK signaling (McQuary et al. 2016); (3) modulation of AMPK (Apfeld et al. 2004; Greer et al. 2007) and ARGK-1 (McQuary et al. 2016) in C. elegans can affect lifespan and stress resistance; and (4) regulation of lifespan by insulin signaling is partially dependent on AMPK signaling in C. elegans (Tullet et al. 2014). As such, we were surprised to find no changes in lifespan upon treatment with a creatine analog. One possible explanation is that, despite the similarity between ARGK-1 and mammalian creatine kinase, the enzymes substrates differ. Biochemical analysis suggests that ARGK-1 uses arginine instead of creatine as a substrate to recharge ADP (Fraga et al. 2015). Additionally, it has been postulated that the biochemical characterization of C. elegans metabolites may have misidentified creatine, and that creatine is not relevant to C. elegans physiology (Witting et al. 2018). Given these caveats, it may not be surprising that -GPA does not alter Caenorhabditis lifespan.