Anti-Aging Effect of the Ketone Metabolite β-Hydroxybutyrate in Drosophila Intestinal Stem Cells

Age-related changes in tissue-resident adult stem cells may be closely linked to tissue aging and age-related diseases, such as cancer. β-Hydroxybutyrate is emerging as an important molecule for exhibiting the anti-aging effects of caloric restriction and fasting, which are generally considered to be beneficial for stem cell maintenance and tissue regeneration. The effects of β-hydroxybutyrate on adult stem cells remain largely unknown. Therefore, this study was undertaken to investigate whether β-hydroxybutyrate supplementation exerts beneficial effects on age-related changes in intestinal stem cells that were derived from the Drosophila midgut. Our results indicate that β-hydroxybutyrate inhibits age- and oxidative stress-induced changes in midgut intestinal stem cells, including centrosome amplification (a hallmark of cancers), hyperproliferation, and DNA damage accumulation. Additionally, β-hydroxybutyrate inhibits age- and oxidative stress-induced heterochromatin instability in enterocytes, an intestinal stem cells niche cells. Our results suggest that β-hydroxybutyrate exerts both intrinsic as well as extrinsic influence in order to maintain stem cell homeostasis.


Introduction
Adult stem cells play a key role in tissue homeostasis and regeneration based on their ability to sustain self-renewal and produce differentiated cells [1][2][3][4]. Age-related changes in adult stem cells are closely involved with tissue aging and age-related diseases, including cancer [5][6][7][8][9]. It is well documented that the microenvironmental niche affects age-related changes in adult stem cells, which is a heterogeneous cell population that surrounds the stem cells [9,10]. Hence, studies need to focus on stem cells and their microenvironments in order to elucidate the mechanisms that slow down or recover age-related changes in adult stem cells.
Organismal diet is emerging as an important regulator of adult stem cell function [11]. Caloric restriction and fasting are commonly associated with extended lifespan, delayed onset of age-related diseases, and reduced cancer incidence, and they are generally beneficial for stem cell maintenance and tissue regeneration [12,13]. Recently, the ketone body β-hydroxybutyrate (β-HB) has emerged as an important molecule for imparting the anti-aging effects of caloric restriction and fasting [14].
Over the last decade, ketone bodies (including β-HB) have been studied for their beneficial outcomes in age-related diseases, such as neurodegenerative disorders and cancer [15,16]. Ketone bodies are small molecules that are synthesized in the liver from fats during fasting, prolonged exercise, or under conditions of restricted carbohydrate supply [17,18]. β-HB is first converted to

Inhibitory Effect of β-HB on Age-and Oxidative Stress-Related Centrosome Amplification in Drosophila Midgut ISCs
Age-and oxidative stress-related centrosome amplification with supernumerary centrosomes has been reported in ISCs. [30,47]. In this study, we investigated the effect of β-HB on age-related centrosome amplification in ISCs of the gut from esg > green fluorescent protein (GFP) flies, which marks only ISC/EB [2]. ISCs were stained with anti-γ-tubulin (a centrosome marker), anti-PH3 (a marker of mitotic ISCs), and anti-GFP (a marker of ISCs/EBs), and the number of cells displaying centrosome amplification was assessed. Supernumerary centrosomes were observed in 5.69% of mitotic ISCs in 45-day-old esg > GFP flies ( Figure 1A(c-c"'),C) and in 7.41% 10-day-old esg > GFP + Cat n1 flies ( Figure 1A(e-e"'),C), a model of intrinsic oxidative stress (Choi et al., 2008) [7], as compared to 1.05% in 10-day-old esg > GFP flies ( Figure 1A(a-a"'),C). The number of mitotic ISCs with supernumerary centrosomes per gut was 2.47 in 45-day-old esg > GFP flies and 7.33 in 10-day-old esg > GFP + Cat n1 flies, as compared to 0.09 in 10-day-old esg > GFP flies ( Figure 1D). These results indicate that, with aging and subsequent to oxidative stress exposure, there is an increase in centrosome amplification in midgut ISCs. Interestingly, β-HB treatment reduces the age-and oxidative stress-related increase in the number of PH3-positive cells ( Figure 1B). β-HB treatment reduces age-and oxidative stress-related increase of supernumerary centrosomes in 3.64% of mitotic ISCs in 45-day-old esg > GFP flies ( Figure 1A(d-d"'),C) and in 5.01% of mitotic ISCs in 10-day-old esg > GFP + Cat n1 flies ( Figure 1A(f-f"'),C). The number of mitotic ISCs with supernumerary centrosomes per gut was reduced by 1.19 in 45-day-old esg > GFP flies, and by 4.08 in 10-day-old esg > GFP + Cat n1 flies, whereas no change was observed in 10-day-old esg > GFP flies ( Figure 1D). We applied PQ treatment as extrinsic oxidative stress to confirm whether β-HB inhibits oxidative stress-induced centrosome amplification in the midgut. Briefly, three-day-old esg > GFP flies with or without 2 mM β-HB treatment for six days were treated with 10 mM paraquat (PQ) for 18 h. Mitotic ISCs (9 to 75.8) and mitotic ISCs with supernumerary centrosomes (1.06% to 8.18%) were both observed to increase in the PQ-treated esg > GFP flies ( Figure 1A(g-g"'),B,C), as compared to control flies ( Figure 1A(a-a"'),B,C). Furthermore, there was an increase in the number of mitotic ISCs with supernumerary centrosomes per after PQ treatment (0.09 to 6.2; Figure 1D). However, in β-HB pre-treated esg > GFP flies, the number of PH3-positive cell (75.8 to 57.06), mitotic ISCs with supernumerary centrosomes (8.18% to 6.13%), and the number of mitotic ISCs with supernumerary centrosomes per gut (6.2 to 3.5) were decreased after PQ treatment ( Figure 1A(h-h"'),B,C). The result from esg > GFP flies indicate that β-HB can reduce age-and oxidative stress-induced centrosome amplification in adult intestinal stem cells in vivo.
In addition, we confirmed the inhibitory effect of β-HB on age-related centrosome amplification in wild type fly, as shown in Figure S1 and the Legend in Supplementary Materials ( Figure S1). These results indicate that β-HB strongly suppresses age-and oxidative stress-induced centrosome amplification in Drosophila midgut ISCs.

Inhibitory Effect of β-HB on Age-Related Increases in DNA Damage Accumulation in Midgut ISCs
Centrosome amplification is associated with cell cycle arrest due to DNA damage, particularly during the G2-M phase [47,48]. In this study, we assessed the correlation between DNA damage and centrosome amplification, while considering the inhibitory effects of β-HB. Previous studies have reported the age-and oxidative stress-induced increases of DNA damage accumulation in Drosophila midgut ISCs [28,29]. Based on these studies, we undertook examining whether β-HB treatment reduces the age-and oxidative stress-induced DNA damage accumulation. This study confirmed the presence of age-and oxidative stress-induced increases of DNA damage accumulation in midgut ISCs after aging and oxidative stress exposure. Briefly, the cells were stained with anti-γ-H2AvD (a DNA damage marker) and anti-GFP (diploid cells marker, for both ISCs and EBs). The level of γH2AvD fluorescence in esg-positive cells increased in 45-day-old esg > GFP flies (13.31, Figure 2A(c-c"),B),in 10-day-old esg > GFP + Cat n1 flies (7.68, Figure 2A(e-e"),B), and in PQ-treated esg > GFP flies (4.03, Figure 2A(g-g"),B), as compared to 10-day-old esg > GFP flies (2.12, Figure 2A(a-a"),B). As expected, β-HB treatment resulted in reduced DNA damage accumulation in esg-positive cells of 45-day-old esg > GFP flies (13.31 to 5.97, Figure 2A(d-d"),B), 10-day-old esg > GFP + Cat n1 flies (7.68 to 2.15, Figure 2A(f-f"),B), and PQ-treated esg > GFP flies (7.29 to 3.28, Figure 2A(h-h"),B), as compared to the non-treated flies (Figure 2A(c-c", e-e", g-g"),B). These results indicate that β-HB treatment decreased age-and oxidative stress-related increases of DNA damage accumulation in ISCs.
centrosomes per after PQ treatment (0.09 to 6.2; Figure 1D). However, in β-HB pre-treated esg > GFP flies, the number of PH3-positive cell (75.8 to 57.06), mitotic ISCs with supernumerary centrosomes (8.18% to 6.13%), and the number of mitotic ISCs with supernumerary centrosomes per gut (6.2 to 3.5) were decreased after PQ treatment ( Figure 1A(h-h'''),B,C). The result from esg > GFP flies indicate that β-HB can reduce age-and oxidative stress-induced centrosome amplification in adult intestinal stem cells in vivo.
, f-f'') 2 mM β-HB feeding for seven days, were stained with anti-γH2AvD (red), anti-GFP (green), and DAPI (blue). Ten-day-old esg>GFP flies, without (g-g") or with (h-h") 2 mM β-HB feeding for six days, were treated with 10 mM PQ in standard media for 20 h, after which their guts were stained with anti-γH2AvD (red), anti-GFP (green), and DAPI (blue). a', b', c', d', e', f', g', and h' indicate enlarged GFP stained images. a", b", c", d", e", f", g", and h" indicate enlarged γH2AvD stained images. Original magnification is 400×. (B) Graph showing the average fluorescence intensity of γH2AvD in GFP-positive cells in 10-day-old esg > GFP, 45-day-old esg > GFP, 10-day-old esg > GFP + Cat n1 mutant, and 10-day-old PQ-treated esg > GFP flies, with or without β-HB feeding for seven days. N is the number of observed guts and n is the number of observed cells. The error bar represents standard error. p-values were calculated using Student's t-test. n.s. indicates not significant (p > 0.05).

Discussion
In the present study, we report that β-HB, a key molecule that is involved in ketone body signaling, has an inhibitory effect on adult stem cell aging in the Drosophila midgut.
The key results of this study are: (1) β-HB inhibits age-and oxidative stress-induced increases of centrosome amplification in midgut ISCs (centrosomes amplification are reported to be an early event in tumorigenesis and senescence), (2) β-HB inhibits age-and oxidative stress-induced DNA damage accumulation in midgut ISCs and progenitors, and (3) β-HB inhibits age-and oxidative stress-induced increases of heterochromatin instability in midgut ECs, ISC niche cells.
Our results further indicate that β-HB treatment reduces the age-and oxidative stress-induced DNA damage accumulation in midgut ISCs and progenitors, when considering the changes that were obtained in the fluorescence intensity of γH2AvD (the phosphorylated Drosophila histone variant of H2A on Ser137, homologous to mammalian γH2AX) (Figure 2). GammaH2AX is a well-established marker of DNA damage [57], and an irradiation-induced γH2AvD increase in ISCs indicates that the γH2AvD signal in ISCs does indicate the presence of DNA damage in Drosophila [58]. We have previously reported that an increase in γH2AvD is a key feature of Drosophila ISC aging and the changes in γH2AvD is closely associated with the age-related accumulation of 8-oxo-2 -deoxyguanosine (a well-known product of ROS-induced DNA damage) [28]. β-HB induces an increase in the levels of the ROS scavengers, viz., catalase, and SOD [59]. More recently, β-HB has been shown to prevent cell senescence and exert anti-aging effects through the upregulation of Oct4 and Lamin B1, which are key factors acting against DNA damage [14]. In the current study, we show an additional benefit of β-HB, in its ability to protect against DNA damage accumulation in stem cells.
Our results further indicate that β-HB treatment recovers the age-and oxidative stress-induced loss of heterochromatin stability consequent to the loss and dispersion of H3K9me3 and HP1 in midgut ECs (Figure 3). It is well known that ECs surround ISCs [2,3] and they are a major source of niche signal on ISC self-renewal [60]. ECs are constantly exposed to external stresses, such as injury, infection, or damaged conditions, resulting in ECs death, which is a major cause of accelerated ISC proliferation [25,31,61,62]. A previous study reported that age-related loss of heterochromatin stability in differentiated ECs is associated with an increase of age-related phenotypes of ISC (hyperproliferation and DNA damage accumulation) through apoptotic death, as a niche aging [49]. These observations indicate that β-HB acts as an anti-aging agent in the adult fly midgut ISCs directly via the reduction of centrosome amplification and DNA damage accumulation, and it also affects niche survival via increased heterochromatin stability. When considering the previous study results, it is likely that β-HB is intrinsically as well as extrinsically involved in the maintenance of adult ISCs. In a previous study, we also showed that AKT/TOR signaling induces DNA damage accumulation in Drosophila ISCs/EBs [29]. Another study reported that TOR activity in ECs increases with age, and the increased AKT/TOR signaling in ECs is related to the loss of heterochromatin stability in ECs [49]. It is well documented that the inhibition of mTOR is directly implicated in lifespan extension [9,10]. β-HB is known to inhibit mTOR signaling in intestinal cells [63]. Further studies are required to explore whether the inhibitory effects of β-HB on age-related change of ISCs are associated with AKT/TOR signaling. The data of life span in Drosophila are also required to know whether β-HB is really effective on aging in both physiological and pathological conditions.
In summary, the data that were obtained in the current study demonstrate that β-HB reduce age-and oxidative stress-induced centrosome amplification, hyperproliferation, and DNA damage accumulation of ISCs, and the loss of heterochromatin stability in ECs, also regarded as the ISC niche cells in the Drosophila midgut. Our data suggest that β-HB supplementation has the potential to maintain stem cell homeostasis, both intrinsically and extrinsically.

Fly Stock
Fly stocks were maintained at 25 • C on standard food under an approximate 12 h/12 h light/dark cycle. Food consisted of 79.2% water, 1% agar, 7% cornmeal, 2% yeast, 10% sucrose, 0.3% bokinin, and 0.5% propionic acid. In order to avoid larval overpopulation in all vials, 50-60 adult flies per vial were transferred to new food vials every 2-3 days for a period of 50-60 days or longer. The esg-Gal4,UAS-GFP/CyO strain was provided by the Drosophila Genetic Resource Center (DGRC, Kyoto, Japan). The esg > GFP (esg-al4,UAS-GFP/+; +/+) flies were obtained from a cross of Oregon-R males and esg-Gal4,UAS-GFP/CyO females. Oregon-R flies were used as the wild type. The Catalase heterozygous mutant flies (Cat n1 mutant, Bloomington Drosophila Stock Center), a model of intrinsic oxidative stress, is based on previous reports showing a gene dosage-dependent effect on catalase activity [64]. The results described in this study were obtained using female flies.

Immunochemistry
Intact adult guts were dissected and fixed at room temperature. For anti-green fluorescent protein (GFP) antibody staining, the guts were fixed for 1 h in 4% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA). For anti-γH2AvD and Dl antibody staining, the guts were fixed for 30 min in 4% paraformaldehyde (Electron Microscopy Science, USA), dehydrated for 5 min. in 50, 75, 87.5, and 100% methanol, and rehydrated for 5 min. in 50, 25, and 12.5% methanol in PBST (0.1% Triton X-100 in phosphate-buffered saline) for postfixing. After washing with PBST, the samples were incubated for 1 h with secondary antibodies at 25 • C, washed again in PBST, mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA), and then analyzed using a Zeiss Axioskop 2Plus microscope (Carl Zeiss Inc., Göttingen, Germany). The PH3 + cells were counted in the entire midgut.

β-Hydroxybutyrate Pre-Feeding Assay
Three-day-old esg > GFP or wild type flies treated with 2 mM β-HB for six days [18,22]. After treatment with 10 mM paraquat (PQ, methyl viologen, Sigma-Aldrich) in standard food media for 18-20 h at 25 • C and then guts were analyzed by immunostaining.

Paraquat Feeding Assay
Flies were fed 10 mM PQ in standard media for in standard food media for 18-20 h at 25 • C and then guts were analyzed by immunostaining.

Quantitative Analysis
The number of PH3-positive cells in the whole gut was counted to quantitatively analyze PH3-positive cells. For quantitative analysis of centrosome amplification, we determined the number of γ-tubulin stained spots per PH3-positive cell in the whole midguts. To quantitatively analyze the percentage of H3K9me3 and HP1, individual enterocytes in the posterior midgut from obtained images were cropped to individual image files, and serially analyzed. Each value was obtained as the percentage in a single midgut, and the results are given as the mean of each percentage of midguts. The quantified data are expressed as the mean ± SE. Significant differences were identified while using Student's t-test. Sigma Plot 10.0 (Systat Software Inc., San Jose, CA, USA) was used for analysis of standard error.

Quantification of γH2AvD Fluorescence Levels (Means)
Adobe Photoshop CS6 extended software (Abode System Inc., San Jose, CA, USA) was applied to quantify integrated fluorescence intensities from independent samples for each condition and genotype analyzed. The fluorescence level of foci was measured within the nucleus, whose boundary was