Endocrine Abstracts

Compartmentalised regulation of NAD⁺ is intrinsic to key processes including cellular metabolism, and tissue differentiation. Using NAD⁺, the post-translational modification ADP-ribose is applied to proteins altering their biological activity. Our recent work established ADP-ribosylation in skeletal muscle to exist beyond the nuclear compartment and impact the critical steroid hormone class, glucocorticoids. Here, we hypothesised that like NAD⁺, ADP-ribosylation is compartmentalised and its interplay with glucocorticoids also occurs in a subcellular-specific manner. ADP-ribosylation occurs in two variants, a single ADP-ribose subunit (mono-ADP-ribosylation) or branched multiple ADP-ribose units (poly-ADP-ribosylation). These occur independently and are distinct, but both are poorly understood. In whole muscle cells (C2C12s), we show the synthetic glucocorticoid, dexamethasone (1μM) to inhibit poly-ADP-ribose levels (0.61 fold change ± 0.13 sem, P < 0.01; n = 8) and conversely, induce mono-ADP-ribosylation (1.34 ± 0.20, P < 0.0001 n = 6). In parallel, dexamethasone increases cellular NAD⁺ significantly (20% increase over vehicle control, P < 0.02; n = 8). This demonstrates the rate-limiting nature of NAD⁺ and poly-ADP-ribosylation, but not the mono-variant. Subcellular fractionation of C2C12s reveals poly-ADP-ribosylation is absent from the cytoplasm but detected in microsomes, mitochondria, and nuclei. We find Mono-ADP-ribosylation in all compartments examined. Furthermore, with immunoblotting, poly-ADP-ribose is decreased in mitochondria (0.75 ± 0.08) and nucleus (0.85 ± 0.07) and increased in microsomes in the presence of dexamethasone (P < 0.05; n = 3). Interestingly, mono-ADP-ribosylation is significantly elevated in all the fractionated organelles in response to dexamethasone (nucleus: 1.61 ± 0.30; mitochondria: 2.37 ± 0.68; microsomes: 3.36 ± 1.18; cytoplasm: 3.28 ± 1.14). These findings not only highlight the struggle between cellular compartments for NAD⁺ but also the capacity to produce poly- and mono-ADP-ribosylation. Moreover, these two post-translational modifications shift in a compartmentalised specific manner as a response to exposure to glucocorticoids. Our findings suggest ADP-ribosylation is a common but poorly understood mechanism involved in affecting steroid hormone signalling in skeletal muscle.