Int. J. Mol. Sci. 2018, 19, x; doi: FOR PEER REVIEW www.mdpi.com/journal/ijms
1 Review
2 Drosophila gut—a nexus between dietary restriction and lifespan
3 Ting Lian1,†, Qi Wu1,† ,Brian A. Hodge2, Guixiang Yu1, Mingyao Yang1,*
4 1 Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Chengdu
5 611130, P. R. China
6 2 Buck Institute for Research on Aging, 8001 Redwood Blvd., Novato, CA, 94947, USA
7 * Correspondence: yangmingyao@sicau.edu.cn; Tel.: +86 28 028‐86290991
8 † These authors have contributed equally to this work
9 Received: date; Accepted: date; Published: date
10
11 Abstract: Aging is often defined as the accumulation of damage at the molecular
12 and cellular levels that overtime result in marked physiological impairments
13 throughout the organism. Dietary restriction (DR) has been recognized as one of
14 the strongest lifespan extending therapies observed in a wide array of organisms.
15 Recent studies aimed at elucidating how DR promotes healthy aging have
16 demonstrated a vital role of the digestive track in mediating the beneficial effects
17 of DR. Here, we review how dietary restriction influences gut metabolic
18 homeostasis and immune function. Our discussion is focused on studies in the
19 Drosophila digestive track where we describe in detail the potential mechanisms in
20 which DR enhances maintenance of intestinal epithelial barrier, up‐regulates lipid
21 metabolic processes, and improves how the gut deals with damage or stress. We
22 also examine evidence of a tissue‐tissue crosstalk between the gut and neighboring
23 organs including the brain and fat body. Taken together, we argue that the gut
24 plays a critical role in DR mediated lifespan extension.
25 Keywords: Drosophila; gut; aging; dietary restriction; intestinal epithelia barrier
26
27 1. Introduction
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Aging is often described as a lifelong 28 process in which a variety of damage
29 accumulates overtime in molecules, cells and tissues, thereby resulting in a decline
30 in physiological functions [1‐3]. It is accompanied with a loss in proliferative
31 homeostasis and regenerative capacity in high‐turnover tissues, as stem cell pools
32 become exhausted. Such is so in the ability of the gut to repair itself into old age [4‐
33 6]. Thus, the dysfunction of molecules, cells as well as tissues result in a range of
34 aging‐related diseases such as cancer, cardiovascular diseases [7, 8]. The intestinal
35 epithelium forms a selective barrier to allow nutrient absorption while keeping the
36 microbiota within the lumen of the gut. To maintain proper gut homeostasis, the
37 intestinal epithelia cells mount frequent and required immune responses against
38 potentially harmful entities (if not maintained within the gut) such as pathogenic
39 microorganisms, dietary antigens, and environmental toxins. Simultaneously, the
40 intestinal epithelium is also involved in mutually beneficial interactions with
41 commensal life‐forms that shape the host immune system, providing the essential
42 metabolic functions and permitting the absorption of nutrients, ions, and water [9,
43 10]. In aging animals the intestine suffers structural and functional impairments,
44 thereby diminishing intestinal barrier function [11‐14], which can promote other
45 aging related diseases such as cancer, inflammatory bowel disease (IBD), ulcerative
46 colitis, and Crohn’s disease [15].
47 Dietary restriction (DR) has been demonstrated as one of the most robust
48 interventions to extend lifespan across single‐celled organisms, invertebrates, and
49 vertebrate animals [16]. The term DR includes a broad range of interventions such
50 as short‐term starvation, periodic fasting, fasting‐mimetic diets, intermittent fasting,
51 normo‐caloric diets with planned deficiencies (in particular macronutrients) and
52 time‐restricted feeding [17]. DR exerts its salutary effects by regulating
53 evolutionarily conserved signaling pathways including major nutrient‐sense
54 pathways (insulin signaling and mTOR), stress‐related pathways such as c‐Jun N55
terminal kinase (JNK) signaling, and pathways involved in intestinal proliferation
56 such as JAK/STAT signaling [10, 18‐20]. Furthermore, the long‐term maintenance of
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organismal 57 homeostasis is mediated by DR is dependent on interactions between
58 organs systems [6, 21]. Recently the Drosophila intestine has emerged as an appealing
59 model to explore tissue dynamics (i.e. regenerative capacity) with aging because of
60 its genetic, morphological and functional simplicity and experimental accessibility
61 by using the sophisticated genetic tools as well as the high structure similarity and
62 evolutionary conservation of intestinal regeneration with human [22, 23]. In this
63 review, we focus on how DR effects the Drosophila gut, summarize the recent
64 advances in our understanding of intestinal homeostasis throughout aging, and its
65 interaction in mediating DR benefits of lifespan and organismal health.
66 2. Gut function during DR-induced longevity
67 2.1. Epithelial homeostasis with aging
68 The intestinal epithelium provides the selectively permeable barrier that
69 functions to absorb nutrients while preventing the uptake of toxins and microbial
70 contamination [24]. This barrier is maintained by self‐renewing intestinal stem cells
71 (ISCs) that sense damage and promote intestinal regeneration. ISCs constitute the
72 majority of cells capable of mitoses in Drosophila midgut epithelia, and respond to
73 an array of different environmental stressors and nutritional conditions. The ISCs
74 thereby preserve the integrity of the intestinal barrier by adjusting epithelium size
75 in response to changing stresses and dietary conditions [25]. In young flies, or in
76 states of low stress, ISCs are found in a ‘quiescent state’ as their proliferation is
77 relatively slow‐ or non‐existent [26] that allows the replacement of intestinal
78 epithelium with symmetric division (one ISC divides into two ISC clones). This self79
renew division allows the stem cell pool to be scaled according the needs of gut
80 tissue [27]. Throughout aging, environmental stress and damage results in an
81 accelerated proliferation of ISCs with asymmetric division, which is often referred
82 to as the “proliferation state” [28]. ISCs generate daughter cells, enteroblasts (EBs).
83 Unlike the mammalian intestinal crypts, in fly epithelium, ISCs reside in visceral
84 muscle, while EBs localize apically to be the mother stem cells and 90% of EBs
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differentiate into polyploidy 85 EBs to form the intestinal epithelium, 10% of EBs
86 appear to differentiate into either secretory enteroendocrine cells (EEs, small,
87 diploid) or absorptive enterocytes (ECs, large, polyploid) [29, 30]. ISCs Throughout
88 aging, ISCs hyperproliferate and drive intestinal dysplasia [31]. In addition to
89 intestinal dysplasia, a common hallmark of the aging gut is a progressive loss of
90 barrier function such that older guts lose the ability to selectively regulate nutrients
91 and maintain the microbiota in the intestinal lumen [4, 12, 32‐34]. The intestines of
92 elderly flies display an increase of stem cell proliferation, a loss of terminal
93 differentiation of progenitor cells, activation of inflammatory pathways, and
94 increased intestinal permeability [34, 35]. This loss of intestinal homeostasis is
95 considered a hallmark of aging in both flies and humans, and is associated with the
96 progression of other aging‐related diseases [36‐38]. In flies, intestinal epithelial
97 barrier dysfunction has served as a predictor of mortality as flies that have
98 permeable guts display a decrease in longevity [12, 32, 39]. Our current
99 understanding of the underlying molecular mechanisms that regulate intestinal
100 epithelia maintenance and the age‐associated loss of barrier function is limited and
101 is an active field of study. This section may be divided by subheadings. It should
102 provide a concise and precise description of the experimental results, their
103 interpretation as well as the experimental conclusions that can be drawn.
104 2.1.1. DR and DR mimetics improve gut epithelial homeostasis
105 DR and treatment with DR mimetics such as Rapamycin, 2,5‐dimethyl‐celecoxib
106 (DMC), and metformin have been shown to promote gut epithelial homeostasis with
107 aging [12, 33, 34, 40], suggesting that lifespan extension by these therapies may be
108 mediated in part by beneficial effects on gut health. Upon DR (and DR mimetics),
109 flies display intestinal barrier loss at much slower rates compared to flies reared on
110 control or nutrient rich diets. Since Rera et al developed a noninvasive assay to
111 determine individual fly intestinal integrity, this assay has been used in fly intestinal
112 experiments [4, 32, 41]. In this assay, flies with loss of intestine barrier integrity are
113 considered as “Smurf” flies. DR and DR mimetics decrease the Smurf increase rate
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[12]. Secondly, intestine size is diet dependent 114 [27, 42]. Flies fed on rich nutrient diet
115 show the increase of total intestine cells due to the misdifferentiation of ISCs which
116 results in the increase of intestine gross size. The accumulation of ISCs and mis117
differentiated daughter cells is significant decreased in old flies on DR or DR
118 mimetics. Among which, the number of esg (transcription factor escargot, ISCs and
119 EBs specific marker) and Delta (the Notch ligand, specific expressed in ISCs) positive
120 cells are significant decreased. And the decrease of mitosis marker phosphorylated
121 histone H3 (PH3) positive cells is also observed, which shows the decrease of ISCs
122 proliferation [4, 34, 41]. In addition, intercellular occluding junctions also show the
123 critical role in maintaining intestinal barrier integrity such as tricellular junctions
124 (TCJs), which is disrupted with aging. Giotactin (Gli) is localized to TCJ in Drosophila.
125 Renisk‐Docampo et al. recently demonstrated that Gli largely absent from TCJ in old
126 flies midguts, and depletion of Gli in ECs results in the impairment of intestinal
127 regeneration thereby accelerating loss of intestinal barrier integrity while DR delays
128 the changes of Gli localization at TCJs in old flies [4, 10]. So DR shows the
129 improvement of regenerative capacity by enhancing the expression of Gli at late age.
130 Thus, DR and DR mimetics are able to maintain the regenerative capacity of
131 intestinal stem cells population and promotes flies have greater response of lifespan
132 to DR and DR mimetics.
133 2.1.2. Pathways
134 Aging related intestinal epithelial barrier dysfunction contributes to functional
135 degeneration including the disorder of intestinal immunity homeostasis across
136 invertebrates to humans and the incidence of cancer such as colorectal cancer [35,
137 43]. ISC regenerative declines with aging have been shown to be regulated by both
138 cell intrinsic as well as external environmental challenges [4, 42]. Recent studies have
139 demonstrated the involvement of a number of signaling pathways that regulate stem
140 cell stress tolerance and repair. The precise coordination of protective and damage
141 control mechanisms remain to be established. Here we summarize the current
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signaling pathways that control ISC proliferation, 142 differentiation, and the function
143 in the context of DR mediated longevity.
144 ISCs sense damage and proliferate throughout life, while with aging they
145 ultimately lose regenerative capacity that induces the increase of ISC proliferation
146 combined with the accumulation of mis‐differentiated daughter cells [4, 10]. DR and
147 DR mimetics delay the over‐proliferation of ISCs in old flies. The nutrient signaling
148 pathways, insulin (IIS) and target of Rapamycin (TOR) pathways communicate
149 nutrient and energy levels to downstream transcriptional regulators that control ISC
150 function (Figure 1).
151
152 Figure 1. DR and DR mimetics improve gut epithelial function. In fly gut, the
153 epithelial homeostasis is disrupted with aging, causing dysplasia. Dietary
154 restriction or its mimetics delays this process through various pathways including
155 IIS signaling, TOR pathway, JNK, JAK/STAT pathway, IMD, Ras/MAPK pathways.
156
157 DR reduces signaling through the IIS pathway which is required for ISC
158 proliferation [44]. Genetic activation of the IIS pathway by expressing INR has been
159 shown to induce intestinal dysplasia [26, 45]. Limiting IIS signaling activity extends
160 lifespan in flies. These flies have reduced intestinal dysplasia including the
161 reduction of pH3+ cells, intestinal BrdU incorporation, and the loss of intestinal
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epithelial architecture. Reducing the IIS activity 162 in the ISC lineage shortens flies
163 lifespan by impairing the ISC proliferation. In addition, transcription factor foxo is
164 repressed by IIS and required for lifespan extension at limited IIS activity. Loss of
165 foxo in mouse hematopoietic stem cell (HSCs) results in the increase of HSC
166 proliferation and the elevation of reactive oxygen species (ROS) level, consequently
167 leading to the reduction of HSC pool regenerative ability [46‐48]. However, if
168 selected over‐expressing the targets of Foxo such as jafrac1 (a peroxiredoxin that
169 detoxifies ROS) and hsp68 (a heat shock protein) is able to limit the effects of IIS in
170 the ISC lineage, delay the age‐related intestinal epithelia dysfunction. In addition to
171 Foxo‐mediated cell‐autonomous ISC proliferation mechanism, IIS also
172 nonautonomously regulates ISC proliferation, that is, InR is necessary to the EBs
173 differentiation, and modest differentiation of EBs allows further ISCs division then
174 suppresses the aging phenotype‐intestinal dysplasia in reduced IIS level flies [49].
175 These studies indicate that fly lifespan is extended when intestinal stem cell
176 proliferation is reduced but not completely inhibited, and thus highlight a key
177 balance in promoting intestinal homeostasis. Likewise, age‐related intestinal
178 dysfunction is reported to be caused by the activation of Jun‐N‐terminal Kinase
179 (JNK) signaling pathway which accelerates ISCs activity and ultimately results in
180 over‐proliferation [31, 35]. Reduction of JNK signaling activity in ISCs promotes
181 lifespan extension in flies similar with reduction of IIS signaling [26].
182 The nutrient responsive TOR signaling cascade has been widely demonstrated
183 as potent regulator of the aging process, as genetic or pharmacological inhibition of
184 TOR has been shown to extend lifespan in a number of animal models [34, 50‐52].
185 Additionally, DR‐mediated health benefits and longevity have been attributed in
186 part to a decrease in TOR signaling activity. TOR, which is a serine/threonine protein
187 kinase, integrates growth cues downstream of PI3K and AKT signaling cascades,
188 and regulates many downstream biological processes including mRNA translation,
189 cellular growth, stress resistance, mitochondria biogenesis, autophagy, and stem cell
190 function [53, 54]. In flies, TOR is involved in maintaining stem cell identity, and
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regulating differentiation of ISCs in 191 a nutrient‐dependent manner. TOR signaling
192 plays a critical role for maintaining the stem cell pool by maintaining stem cell
193 identity as well as ISC proliferation and promoting the symmetric differentiation of
194 EBs into ECs and EEs. TOR activity is lower in ISCs than EBs since the TSC2 protein
195 which acts as a suppressor of TOR is highly expressed in ISCs but not in EBs.
196 Inhibition of TSC2 downstream of the Notch signaling pathway in EBs, activates
197 TOR and thus promotes the commitment of EBs into the ECs fate [44, 55]. These
198 observations are consistent with studies in mice that demonstrate lower activity of
199 mTOR is detected in Paneth cells (which is the ISC‐supporting cells) under DR,
200 which regulate ISC regeneration through mTOR by sensing the organismal
201 nutritional status [56]. While recent report shows mTOR activity is up‐regulated in
202 ISCs upon DR, which forces ISC proliferation. . Rapamycin treatment which
203 represses TOR activity acts as a DR mimetic by blocking ISC expansion in mice fed
204 in DR diet with suitable dose [57]. Thus, drugs like DR mimetics – rapamycin may
205 attribute opposite effects on different cell types. And the concise molecular
206 regulating mechanisms of TOR in ISC proliferation and ISC lineage differentiation
207 need to be explored further.
208 The intestinal epithelium is continually challenged by pathogenic bacteria as
209 well as the commensal microbiota which can influence intestinal homeostasis,
210 immune stress responses, and the regenerative activity of the epithelial tissue. To
211 combat potentially harmful pathogens, the intestinal epithelium will respond to
212 damage by increasing the expression of antimicrobial peptides (AMPs). AMPs are
213 mainly regulated by Toll and Immune Deficiency (IMD) innate immune pathways
214 [58, 59]. In addition, AMPs can be directly activated by the transcription factors
215 Drosophila Forkhead boxO (dFoxo) or Forkhead (Fkh). Foxo and Fkh are directly
216 repressed by IIS and TOR signaling [60], so in the absence of IIS/TOR signaling
217 AMPs can be activated by Foxo and Fkh upon DR or DR mimetics[34, 41]. In the fly
218 midgut, AMPs are regulated by IMD and the Janus kinase‐signal transducers and
219 activators of transcription (JAK‐STAT) pathways [61] and caudal, the negative
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transcriptional regulator [62] not Toll signaling. 220 When ubiquitously or gut‐specific
221 over‐expressing one of AMPs–Drosocin (Dro), flies show lifespan extension
222 accompanied with the reduction of IMD and JAK‐STAT regulating AMPs in the flies
223 midgut as well as the Jun N‐terminal kinase (JNK) and Epidermal growth factor
224 receptor (EGFR), which is required for intestinal regeneration and ISC pool
225 homeostasis [63]. These pathways are usually regarded as the makers of intestinal
226 homeostasis because of their elevated activity with aging or response to bacteria
227 challenge [64, 65]. Changes in the composition of microbiota can trigger chronic JNK
228 and JAK‐STAT signaling activity with aging, which in turn promotes ISCs over229
proliferation, resulting in intestinal epithelia dysplasia [35]. Furthermore, Loch and
230 colleagues also observed the gut permeability is significantly decreased in over231
expressing Dro flies with aging. The improved intestinal barrier is also observed in
232 DR flies, eliciting the crosstalk of nutrient, innate immunity, intestinal homeostasis,
233 and aging [63].
234 2.2. Intestinal lipid homeostasis
235 Maintaining proper lipid metabolic homeostasis is central to organismal health.
236 Disrupting lipid synthesis and/or breakdown is a major risk factor for metabolic
237 diseases such as obesity, type‐2 diabetes, and cardiovascular diseases [66, 67]. Under
238 normal conditions, lipid homeostasis is maintained by absorption of dietary lipids
239 through the intestinal epithelium into the circulation where peripheral tissues can
240 either store excess lipids or metabolism them for energy. The process of dietary lipid
241 absorption begins with the breakdown of lipids (including triacylglycerol (TAG) and
242 cholesterol esters) into free‐fatty acids (FFAs), monoacylglycerols, and free sterols in
243 the intestinal lumen [68, 69]. FFAs are absorbed by intestinal cells (ECs) and
244 resynthesized into TAGs, then packaged into lipoprotein particles together with
245 cholesterol, cholesterol esters, and carrier proteins [19]. These lipoprotein particles
246 are trafficked to peripheral tissues. These lipid can be either used by cells for energy
247 or deposited in storage tissues such as fat body and intestine [67]. Thus, as stated
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above, the misdifferentiation of EBs to ECs 248 with aging will disrupt the lipid
249 metabolism in intestine, thereby influencing organism health.
250 2.2.1. DR maintains intestinal lipid homeostasis
251 Accumulating studies have characterized the molecular mechanisms of lipid
252 uptake, synthesis, catabolism and mobilization which takes place in the intestine [19,
253 70‐73] (Figure 2).
254
255 Figure 2. DR maintains the intestinal lipid metabolisms. Dietary restriction or its
256 mimetics promotes the adaptation towards triglycerides usage, increases the lipid
257 accumulation and fat storage in fly gut. This process is mediated by a range of
258 hormones and prohormones. And the endoplasmic reticulum (ER) stress signaling
259 is also involved.
260 In the fly there is a progressive loss in the intestine’s ability to synthesize and
261 store lipids with aging because of the decline of the number of ECs in the intestine
262 resulting in the decrease in the ability to transport lipids or absorb lipids from lumen
263 into ECs. Restoration of intestinal lipid metabolism has been reported to extend
264 lifespan in the fly [74]. DR improves intestinal epithelia barrier function and also
265 promotes a metabolic shift towards enhanced utilization of lipids and increased
266 mitochondria function [73, 75‐77]. DR promotes the conversion of dietary
267 carbohydrates into lipids, increases the synthesis and breakdown of fatty‐acid, and
268 accelerates lipid turnover in flies. Knockdown of the TAG synthesis gene Acc ablates
269 DR‐mediated lifespan extension therefore highlighting the importance of lipid
270 metabolism upon DR, [73, 78].
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Intestinal cells 271 secret various hormones including cholecystokinin (CCK),
272 ghrelin, glucagon‐like peptide‐1 and 2 (GLP1 and 2) [70]. Tachykinin (TK) is a
273 prohormone in midgut EEs [79]. TK encodes 6 mature peptides (TK1‐6) which is
274 expressed in the anterior, middle and posterior midgut [80]. Gut prohormones
275 promote gut contraction and maintenance of gut peristalsis [81, 82]. Recently, Song
276 and colleagues revealed the physiological role of TK in regulating the intestinal lipid
277 homeostasis. This group showed that TK represses lipogenesis in ECs through
278 TKR99D (a G‐protein‐coupled TK receptor in the gut) and protein kinase A (PKA)
279 signaling [70, 83, 84].
280 Moreover, endoplasmic reticulum (ER) stress has been shown to link lipid
281 homeostasis as well as human diseases including diabetes and metabolic syndrome
282 [85, 86]. During ER stress, the transducer IRE regulates ER homeostasis by inducing
283 genes involved in ER biogenesis, protein folding and degradation through
284 dimerizing and splicing XBP1 [87]. Both IRE and XBP1 are required for lipid
285 homeostasis with increased lipogenesis and lipid usage [88‐91]. Recently, the novel
286 role of IRE1/XBP1 ER stress signaling module in ECs is established, that is,
287 regulating the shift towards the increase of intestinal TAG usage upon DR associated
288 with sugarbabe (a Gli‐like zinc‐finger transcription factor) [19], consequently
289 beneficial for lifespan. It suggests that IRE1/XBP1/Sugarbabe signaling mediates the
290 metabolic adaptation of intestinal epithelium upon DR.
291 2.3. DR improves the intestinal oxidative stress resistance
292 DR has been reported as the anti‐aging paradigm in protecting against
293 oxidative stress induced diseases through reducing reactive oxygen species
294 production, increasing antioxidant enzyme activity as wells as increasing the
295 turnover of oxidized macromolecules [92]. Flies’ intestine is thought to be the
296 simpler model to characterize the increased ability of oxidative stress resistance
297 that induced by aging or the oxidants such as paraquat and H2O2 [33, 93‐95].
298 (Figure 3).
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299
300 Figure 3. The improvement of DR against oxidative stress. DR and DR mimetics
301 enhances mitophagy and promotes the elimination of damaged mitochondria
302 DNA through PINK1/Parkin signaling, meanwhile ROS is also reduced. In
303 addition, mitochondria respiratory capacity and biogenesis are also improved with
304 the increase of dPGC‐1.
305 Oxidative stress is increased with aging and age‐related diseases such as cancer,
306 neurodegeneration, cardiovascular disease, and diabetes [92]. Oxidative stress is
307 caused by an imbalance in the rate of reactive oxygen species (ROS) production and
308 detoxification [96]. Higher ROS levels are observed with tissue damage and aging,
309 or can be induced by the administration of exogenous oxidants such as paraquat
310 and/or hydrogen peroxide in flies midgut [97‐99]. Oxidative stress can damage
311 intracellular macromolecules which results in the disruption of protein/gene
312 expression, cellular dysfunction and death. Overtime accumulating damage caused
313 by ROS can accelerate aging and age‐related diseases [100‐102].
314 In Drosophila, mitochondria is the major generator of ROS and complex I is the
315 main source of ROS associated with aging. Damaged mitochondria usually
316 produces excess ROS. Actually, in long‐lived organisms, there are lower ROS levels
317 produced at complex I, and inhibition of ETC complex can be regarded as a DR
318 mimetics [103].Thus, ROS is an attractive candidate for targeting the aging process.
319 Damaged mitochondria can be degraded by autophagy, mitochondria‐targeted
320 selective autophagy is termed as “mitophagy”, which is regulated by PINK1/Parkin
321 pathway. Increase of autophagosomes by DR and DR mimetics promote the
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elimination of damaged 322 mitochondria induced mtDNA oxidative damage,
323 mitochondria free radical and ROS [33, 34, 104, 105]. In addition, DR can activate the
324 expression of dPGC‐1(Drosophila PGC‐1 homolog, our unpublished RNA‐seq data,
325 peroxisome proliferator‐activated receptor‐ᵧ coactivators), which plays a key role in
326 mitochondria biogenesis and respiration [106, 107] in Drosophila and mammals.
327 Overexpression of dPGC‐1 is sufficient to increase the activity of mitochondria in
328 intestinal epithelium, lower ROS level, and delay the accumulation of
329 misdifferentiated ISCs, thereby improving the gut homeostasis and extending
330 lifespan [32]. One recent report demonstrated that when ISCs senses the oxidative
331 stress, TRPA1 and RyR are identified to regulate cytosolic Ca2+ level in ISCs to
332 activate (by src) and amplify (via autocrine Spi‐EGFR signaling) the downstream
333 EGFR‐Ras/MAPK signaling, thereby in turn inducing the ISCs proliferation [102].
334 p38 MAPK signaling has been reported to maintain fly intestinal host defense and
335 metabolic homeostasis especially p38c. In the guts of p38c fly mutant, ROS level is
336 significant decreased upon bacteria infection [95]. Thus, DR might extend flies’
337 lifespan through decreasing the mitochondria free radical which is regulated by
338 mitophagy as well as improving the mitochondria respiration chain activity.
339 2.4 How gut-other organs communication contributes to the benefits of DR
340 DR‐mediated intestine homeostasis is maintained through a range of signals
341 that originate within the intestine but also through autocrine/paracrine signaling
342 from neighboring tissues. So far, a number of previous evidence suggested the
343 communication of gastrointestinal tract (GI tract) and the neighbor is contributable
344 to maintain the homeostasis of DR benefits occurred in the intestine [6, 21]. Here, we
345 briefly review the signal communication between GI tract and neighbor organs
346 including brain and fat body upon DR.
347 2.4.1. Gut‐brain
348 As stated above, lower level of IIS signaling contributes in the DR‐mediated
349 lifespan extension, which is mediated by eight insulin‐like peptides (dilps 1‐8) that
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act on the insulin receptor on peripheral t 350 issues (DInR, insulin/IGF receptor) in flies
351 [108]. Dilps are primarily secreted from insulin producing cells (IPCs) (median
352 neurosecretory cells, MNCs) in the adult brain including dilp1, 2, 3 and 5 [109]. Dilp
353 5 is also produced in adult ovarian follicles and renal tubules, while dilp3 is
354 expressed in the midgut. Other dilps are expressed in larval fat body, embryo
355 mesoderm, adult central nervous system and so on [108]. The role of Dilps in
356 regulating energy homeostasis and organismal development has been elaborated:
357 glucose metabolism by dilp2, lipid storage by dilp6, lipid metabolism by dilp3, and
358 response to DR by dilp5 [110]. These factors are regulated temporally and spatially,
359 and are also nutrient responsive (see review [110, 111]). Only Dilp5 is likely to
360 mediate DR benefits, but it’s indirect or the secondary role upon DR is still
361 discrepant based on the present literatures in this field. Both dilp5 mRNA and
362 protein level are down‐regulated upon yeast diluted DR diet, while loss of dilp5
363 couldn’t diminish the capacity of DR to extend lifespan [112]. However, dilp5
364 mutant shows the normal DR response and also displayed the up‐regulation of dilp2
365 under higher yeast concentration food while up‐regulated dilp3 with relatively
366 lower yeast concentration. These observations suggested the possible role of dilp5
367 in DR‐dependent lifespan extension is indirect through a compensatory
368 transcriptional regulation [113]. And whether the changes of dilp5 in brain upon DR
369 have an effect on gut function needs to be clarified. Dilp2 can regulate lifespan
370 extension with reduced level through the maintenance of ISC proliferation, which
371 might work through trehalose metabolism rather than DR [114]. Because dilp2
372 mRNA level is elevated upon diet with low protein‐to‐carbohydrate ratios [108]. A
373 recent report from Sano [115] demonstrated the CCHamide‐2 (CCHa2) in the
374 intestine regulates Dilps production in the brain by activation of its receptor CCHa2‐
375 R. But whether the level of CCHa2 changes or not upon DR is not clear. In addition
376 to the direct influences on dilps production, other interesting gut secreted signaling
377 is also documented. For example, AMPK activation in the intestine can regulate
378 autophagy in the brain such as the activity of Autophagy-specific gene 1 (Atg1), and
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Atg1 in turn maintains 379 intestine homeostasis[116]. AMPK is also activated under DR
380 and DR mimetics in Drosophila [117]. Taken together the communication between
381 brain and gut at the regulation of Dilps production and other metabolisms response
382 to DR, if any, consequently have an effect on lifespan.
383 2.4.2. Gut‐fat body
384 A number of previous studies have shown the communications of gut and fat
385 body in regulating the systemic homeostasis including lipid metabolism, AMP
386 production as well as the role of fat body in mediating the intestinal actions [6, 19,
387 70]. Firstly, gut shares the lipid storage and metabolism with fat body [70]. Loss of
388 TK in flies EEs increases the lipid level in fat body, excess gut TK level with
389 deprivation of food induces the loss of systemic lipid storage in fat body through
390 inhibiting sterol regulatory element‐binding protein (SREBP)[70]. In addition, high
391 level of neurotensin (NT) in EEs increases the lipid accumulation in fat body with
392 decreasing AMPK activation [118]. Secondly, gut‐fat body control and regulates
393 systemic AMPs production. AMPs are secreted by fat body, which is modulated by
394 gut‐expressed PGRP‐LE. While PGRP‐LE can be repressed by gut‐expressed
395 amidase peptidoglycan‐recognition proteins (PGRPs) including PGRP‐LB and
396 PGRP‐SCs [119, 120]. What is more, fat body signaling also mediates gut actions with
397 aging. Specific loss of lamin‐B in fat body results in the loss of intestinal epithelium
398 regeneration mediated by Imd pathway [121]. Additionally, nutrient‐signaling
399 pathway such as transforming growth factor β (TGF‐β) signaling is also involved in
400 the intestinal ingestion and absorption. Fat body secreted Dawdle (Dw), the TGF‐β
401 ligand, is responsible for the regulation of carbohydrase and lipase level within
402 midgut through Smad2 [122]. TGF‐β level is regulated in different adipose tissues in
403 mice upon energy restriction, but whether DR regulates this pathway or not needs
404 to be investigated further. As we discussed above, AMPK and IMD is up‐regulated
405 under DR and DR mimetics [34, 117], which speculates the regulation of DR benefits
406 in fatty acid accumulation and the improvement in systemic homeostasis.
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407 3. Conclusion
408 Studies in the simplified Drosophila intestine have made significant research
409 progress in demonstrating the molecular mechanisms of nutrient response to
410 organisms’ lifespan. Here we summarized the present knowledge regarding the role
411 of DR in promoting homeostasis of the intestine epithelial barrier, lipid metabolism,
412 and stress responses. Furthermore, the communication between intestine and the
413 neighbor tissues is briefly discussed, suggesting organ‐organ crosstalk may play a
414 role in promoting the beneficial effects of DR on the gut. While we are beginning to
415 unravel the molecular mechanisms that control the different cell populations in the
416 gut, how DR effects these individual processes with age remains to be studied. The
417 Drosophila and mammalian intestine are share many similarities at the molecular and
418 cellular levels. Therefore, a more comprehensive understanding of Drosophila
419 intestinal physiology and pathology in response to aging and different dietary
420 interventions may translate into findings in higher order animals and humans.
421