Sirtuin-1 Regulates Organismal Growth by Altering Feeding Behavior
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© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs239467. doi:10.1242/jcs.239467 RESEARCH ARTICLE Sirtuin-1 regulates organismal growth by altering feeding behavior and intestinal morphology in planarians Benjamin Ziman1,2, Peter Karabinis1,2, Paul Barghouth1,2 and Néstor J. Oviedo1,2,3,* ABSTRACT metabolism at the systemic level is challenging due to the complex Nutrient availability upon feeding leads to an increase in body size in regulatory mechanisms involved, and the fact that downregulation the planarian Schmidtea mediterranea. However, it remains unclear of sirtuin often results in developmental defects and decreased how food consumption integrates with cell division at the organismal lifespan (Dang, 2014; Lemieux et al., 2005; Li et al., 2007; level. Here, we show that the NAD-dependent protein deacetylases McBurney et al., 2003; Mostoslavsky et al., 2006; Vakhrusheva sirtuins are evolutionarily conserved in planarians, and specifically et al., 2008). Despite widespread interest in understanding the demonstrate that the homolog of human sirtuin-1 (SIRT1) (encoded links between metabolism, homeostasis and disease, the by Smed-Sirt-1), regulates organismal growth by impairing both mechanisms by which sirtuin regulates body growth and animal feeding behavior and intestinal morphology. Disruption of Smed-Sirt- behavior are mostly unknown. 1 with RNAi or pharmacological inhibition of Sirtuin-1 leads to reduced To gain insight into sirtuin function in the regulation of body animal growth. Conversely, enhancement of Sirtuin-1 activity with homeostasis, we focus on the planarian flatworm Schmidtea resveratrol accelerates growth. Differences in growth rates were mediterranea, which is an emerging model organism that has the associated with changes in the amount of time taken to locate food unique capability of responding to nutrient availability by either and overall food consumption. Furthermore, Smed-Sirt-1(RNAi) increasing or decreasing body size. Changes in planarian size are animals displayed reduced cell death and increased stem cell noticeable within a few weeks and result from variations in cell proliferation accompanied by impaired expression of intestinal number across the body rather than changes in cellular size (Baguna lineage progenitors and reduced branching of the gut. Taken and Romero, 1981; Oviedo et al., 2003; Takeda et al., 2009; together, our findings indicate that Sirtuin-1 is a crucial metabolic Thommen et al., 2019). Consequently, smaller animals will contain hub capable of controlling animal behavior, tissue renewal and fewer cells than their larger counterparts, but their proportions are morphogenesis of the adult intestine. maintained despite differences in body size. Independently of their size or metabolic input, planarians are constantly renewing KEY WORDS: Sirtuin, Planarians, Stem cells, Neoblasts, differentiated tissues, fueled by a large pool of adult stem cells Metabolism, Growth called neoblasts. The neoblast is the only cell type with capacity to divide during tissue renewal and in response to injury repair INTRODUCTION (Wagner et al., 2011; Zeng et al., 2018). Therefore, the maintenance Sirtuins are a group of highly conserved protein deacetylases that of body proportionality in planarians results from finely balanced require oxidized nicotinamide adenine dinucleotide (NAD+)asa regulation of cell division and cell death that is greatly influenced by cofactor for their enzymatic function. The ratio of NAD+ to reduced the availability of nutrients. Recent research has revealed nicotinamide dinucleotide (NADH) in response to the nutrient state evolutionary conservation in planarians of signaling pathways of cells links sirtuin function to metabolism. Sirtuin proteins are associated with metabolism (such as TOR and insulin signaling) cellular sensors that function in response to metabolic inputs and (Gonzalez-Estevez et al., 2012; Miller and Newmark, 2012; Peiris that regulate stem cells, growth factor signaling, and food intake et al., 2012; Roberts-Galbraith et al., 2016), but it remains unknown (Arul Nambi Rajan et al., 2018; Calvanese et al., 2010; Dietrich whether sirtuins influence the cellular flux that occurs in response to et al., 2010; Fujitsuka et al., 2016; Hisahara et al., 2008; Igarashi tissue turnover and nutrients in planarians. and Guarente, 2016; Li et al., 2007; Martins et al., 2012; Ou et al., Our results show that sirtuins are molecularly conserved in 2011; Prozorovski et al., 2008; Saunders et al., 2010; Velasquez planarians. We discovered that homologs for six of the seven et al., 2011). Recent evidence implicates sirtuins in a myriad of mammalian sirtuins are present in the S. mediterranea genome human conditions including obesity related disease, aging and (Smed-Sirt-1–6). This is intriguing because evolutionarily related cancer, but the mechanistic roles that sirtuins play in these invertebrate organisms such as Caenorhabditis elegans and conditions remain elusive (DeBerardinis et al., 2008; Houtkooper Drosophila melanogaster possess fewer sirtuin orthologs than et al., 2012; Kurylowicz, 2016). Studying the role of sirtuins in mammals and planarians (Frye, 2000; Vassilopoulos et al., 2011). Furthermore, we found that it is possible to modulate sirtuin 1Department of Molecular and Cell Biology, University of California, Merced, function in planarians using RNAi and treatment with CA 95343, USA. 2Quantitative and Systems Biology Graduate Program, University pharmacological compounds used in vertebrate experimental of California, Merced, CA 95343, USA. 3Health Sciences Research Institute, models and humans (Bhatt et al., 2012; Brasnyó et al., 2011; University of California, Merced, CA 95343, USA. Knop et al., 2013; Liu et al., 2014; Nguyen et al., 2009). Disrupting *Author for correspondence ([email protected]) Sirtuin-1 in planarians led to reduced body size, an unexpected increase in stem cell division and a reduction in levels of cell death. N.J.O., 0000-0002-0213-9781 These effects were accompanied by a reduced branching of the gut. Handling Editor: Maria Carmo-Fonseca Our analysis revealed that Sirtuin-1 plays an integral role in feeding Received 20 September 2019; Accepted 19 March 2020 behavior, regulating not only the length of time taken to locate food Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs239467. doi:10.1242/jcs.239467 and begin feeding but also overall food consumption. Taken with double-stranded RNA (dsRNA) over ten days, and animals together, our findings provide evidence for regulation of cellular were fixed 15 days after the first injection (Fig. 1A). We confirmed turnover by Sirtuin-1, and introduce planarians as a tractable that the protocol was effective in knocking down the gene experimental model to investigate regulation of metabolic inputs expression for two weeks without noticeable abnormalities and cellular behavior in the adult body. (Fig. S2A). Sirtuin function is linked to regulation of metabolism; therefore, RESULTS we analyzed how nutrient availability impacted animal size over Sirtuins are evolutionarily conserved, and Sirtuin-1 is time. First, we prolonged the RNAi protocol for about one month in required for organismal growth in the planarian the absence of nutrients and evaluated the effects on the reduction in S. mediterranea animal size (Fig. S2B). These experiments demonstrated that Sirtuins are a group of NAD+-regulated deacetylase enzymes that disturbing Smed-Sirt-1–6 function does not affect the reduction in are highly conserved from yeast to humans (Frye, 2000; Satoh et al., the overall size of planarians, termed ‘degrowth’ (Fig. S2C). 2011). In the genome of S. mediterranea (Rozanski et al., 2019), we Second, animals subjected to RNAi were exposed to nutrients, and identified homologs for six of the seven human sirtuins, Smed-Sirt- their growth was evaluated for almost two months. This protocol 1–6 (Fig. S1A). Although most of the S. mediterranea sirtuins alternated six feedings with RNAi of the individual Smed-Sirt-1–6 appear evolutionarily conserved, the protein encoded by Smed-Sirt- transcripts and was effective in reducing expression for 1 showed the highest molecular similarity to its counterparts in approximately two months (Fig. 1B; Fig. S2D). The analysis humans and other species, sharing 60% amino acid sequence shows that control and Smed-Sirt-2–6(RNAi) animals increased in identity with human sirtuin-1 (also known as SIRT1) in the size at a similar rate, whereas animals subjected to Smed-Sirt- SIRTUIN domain (Fig. S1A–C). 1(RNAi) displayed a 30% reduction in growth (Fig. 1C,D; Fig. S2E). To functionally characterize the role of sirtuins in planarians, Additionally, Smed-Sirt-1 expression was increased upon feeding, we performed RNAi of the individual Smed-Sirt-1–6 transcripts. suggesting a potential role in planarian metabolism (Fig. S2F). We developed an RNAi strategy based on multiple microinjections Based on these observations, we concluded that Smed-Sirt-1 is Fig. 1. Smed-Sirt-1 regulates planarian growth. (A) RNAi treatment schedule showing timing of microinjections with dsRNA of Smed-Sirt-1. Black bars represent injections with Smed-Sirt-1 dsRNA and the red bar represents the time of fixation. (B) RNAi growth schedule, which combined Smed-Sirt-1 dsRNA microinjections (black bars) with liver feeding (green bars). The red bar represents time of fixation. (C) Changes in animal area over the RNAi growth schedule. Changes