Systemic Activin signaling independently regulates sugar homeostasis,cellular metabolism,and pH balance in Drosophila melanogaster

The ability to maintain cellular and physiological metabolic homeostasis is key for the survival of multicellular organisms in changing environmental conditions. However, our understanding of extracellular signaling pathways that modulate metabolic processes remains limited. In this study we show that the Activin-like ligand Dawdle (Daw) is a major regulator of systemic metabolic homeostasis and cellular metabolism in Drosophila. We find that loss of canonical Smad signaling downstream of Daw leads to defects in sugar and systemic pH homeostasis. Although Daw regulates sugar homeostasis by positively influencing insulin release, we find that the effect of Daw on pH balance is independent of its role in insulin signaling and is caused by accumulation of organic acids that are primarily tricarboxylic acid (TCA) cycle intermediates. RNA sequencing reveals that a number of TCA cycle enzymes and nuclear-encoded mitochondrial genes including genes involved in oxidative phosphorylation and β-oxidation are up-regulated in the daw mutants, indicating either a direct or indirect role of Daw in regulating these genes. These findings establish Activin signaling as a major metabolic regulator and uncover a functional link between TGF-β signaling, insulin signaling, and metabolism in Drosophila.Regulation of cellular metabolism and metabolic homeostasis is crucial for maintaining cellular and organismal physiology. Consequently, robust regulatory networks have evolved in most organisms to adapt and maintain a desired metabolic state depending on the environment and/or developmental stage (1, 2). Deciphering how components of these networks function to achieve homeostasis is essential for understanding normal physiology and the underlying mechanisms behind multifaceted disorders like metabolic syndrome and diabetes. Our understanding of these regulatory networks is largely confined to known metabolic pathways like the insulin signaling (IS) pathway, cellular target of rapamycin (TOR) pathway, and neuroendocrine signals. Nevertheless, additional extracellular signaling mechanisms that can regulate cellular and physiological metabolism must exist to allow integration of environmental, physiological, and developmental cues. In accordance with this possibility, involvement of classical developmental pathways in regulating physiological homeostasis has recently gained much attention (3, 4).One such developmental pathway that has been shown to affect several aspects of metabolism, including nutrient and energy homeostasis, is TGF-β signaling (5, 6). TGF-β signaling has recently been proposed to regulate mitochondrial biogenesis in mammals based on the observation that aberrant Activin signaling in mice can lead to changes in energy metabolism and mitochondrial gene expression (7). However, the study used a gene-replacement strategy where the mature domain of Activin-A was replaced with that of Activin-B (Inhiba^BK), thereby changing the relative levels of these two ligands. TGF-β ligands signal through distinct combinations of type I and type II receptors that are specific to different classes of ligands (8). Although both Activin-A and -B can signal redundantly through the type I receptor ALK4, Activin-B can also signal through ALK7 initiating unique biological responses (9). Hence, studies involving the Inhiba^BK mouse fail to distinguish individual contributions of Activin-A and Activin-B in the manifestation of the energy metabolism defects. Additionally, both Activin-A and -B have been implicated in regulation of IS (10). Because IS is known to impinge on both the TOR and AMPK signaling pathways, aberrant Activin signaling can potentially affect mitochondrial metabolism by affecting IS. Parsing out these mechanistic details is crucial for understanding the role of TGF-β/Activin signaling in metabolism and devising strategies to manipulate this pathway for therapeutic gain.In this study, we use Drosophila melanogaster to investigate the role of TGF-β/Activin signaling in regulation of metabolic homeostasis. Drosophila contains a highly conserved TGF-β signaling pathway. However, the number of signaling components in Drosophila is much smaller than vertebrates, allowing easy genetic manipulation of the pathway and reduces complications arising from functional redundancies between signaling molecules (8). Canonical TGF-β/Activin signaling in the fly is mediated by just three ligands, Activinβ (Actβ), Dawdle (Daw) [also called Alp (Activin-like peptide)], and Myoglianin (Myo), that signal through a single type I receptor, Babo, and a single intracellular R-Smad homolog, Smox (11). In addition, considerable parallels exist in the regulation of metabolic homeostasis between Drosophila and mammals providing a powerful genetic system to investigate the role of TGF-β signaling in metabolism and homeostasis (1, 2, 5).Here, we demonstrate that canonical TGF-β/Activin signaling, mediated by the Drosophila Activin homolog Daw, is a central metabolic regulator that impacts both mitochondrial metabolism and IS. Interestingly, we find that although Daw regulates IS by positively regulating release of Drosophila insulin-like peptides (Dilps), the effect of Daw on mitochondrial metabolism is independent of insulin/insulin-like growth factor signaling (IIS) and may be mediated by changes in expression of nuclear-encoded mitochondrial genes.