Abstract:A restricted diet without malnutrition highly effectively counteracts ageing in several species and extends robustly their lifespan. How lifespan is extended remains enigmatic. We utilized tissue-specific gene expression data in Drosophila to identify mechanisms of DR with higher resolution than ever before.
Introduction
Common Effects of DR on Multiple Tissues
c42: Malpighian Tubule
DJ634: Fatbody/Muscle/Gut
ELAV-C155: Neurons
HAND4.2: Heart
MHC: Thoracic Muscle
Myo1A: Gut
NGT: Germline
S106: Fatbody/Gut
HT: Head/Thorax
Tissue-Tissues Interactions
c42
c42 up
c42 down
DJ534
DJ534 up
ELAV-C155 up
ELAV-C155 down
Hand4.2
Hand4.2 up
Hand4.2 down
MHC
MHC up
MHC down
Myo1A
Myo1A up
Myo1A down
NGT
NGT up
NGT down
S106
S106 up
S106 down
Network Effects
Network Functional Enrichment
Discussion
Common Effects
Discrepancy with Previous Report
DR-Associated Genes
Insulin-like Peptides
Glucagon-Homolog
Takeout
Odorant Receptor
Reaper
Transcription Factors
Gamma-Cystationinase
Hormones
Methods
Gene Expression Profiling
Interaction Networks
Tables & Figures
Ageing is an almost ubiquitous phenomenon. Most species age, with a few exceptions. The ageing process appears to be strongly retarded if the diet is limited to an optimal level, a regimen commonly called dietary or calorie/caloric restriction (DR). The lifespan extension by DR was demonstrated in multiple organisms spanning the kingdom of life from fungi to animals, including mammals. This phenomena appears to be an adaptive response which utilizes defined metabolic reprogramming and changes in the gene expression as well as requires discrete genetic pathways to function as certain mutants and strains do not seem to gain any benefit from DR or even exhibit lifespan shortening. Associated to this response to DR is consistently a reduction in fertility and reproduction. This adaptive conserved DR-response which is nearly ubiquitous, similar to the sole existence of ageing, suggests very ancient and fundamental mechanisms to be activated when nutrients become scare. The most logical explanation for the DR effect from a evolutionary perspective is that in times of famine ageing and reproduction are slowed down in order to survive long enough until the environment allows to reproduce again.
Most gene expression studies in lower model organisms were conducted with whole animals and therefore resulted in an averaging of the gene expression changes from multiple tissues. We here utilize gene expression profiles of different body compartments and tissues [Table: Tissues utilized to investigate the DR effect].
First we checked to which extent the transcriptional response to DR in different tissues overlap. We made binary comparisons of eight different tissues with each other and found that the number of genes in the intersections for the upregulated genes as well as downregulated genes, respectively, are higher than expected by chance.
Next we asked which gene categories are transcriptional affected by DR commonly in all tissues. For this we performed a meta-analysis, but had to lower the expression ratio (experimental / control value) in order to increase the sensitivity. RNA editing and alternative splicing were significantly upregulated in all tissues, while cell division was commonly downregulated [Table: Effects of DR common to multiple tissues 1]. After Benjamini correction Juvenile hormone binding was under the top three significant terms [Table: Effects of DR common to multiple tissues 2].
Among the upregulated lifespan-altering genes is Sh (Shaker), which mutation results into premature ageing, while among the downregulated genes is Hsp27 of which over-expression leads to lifespan extension.
We then investigated the changes specific to a particular body compartment/tissue.
Among the upregulated gene categories in the malpighian tubule are immunoglobin-like terms (fs(1)M3, esc and Sin3A.
In the fatbody/muscle/gut DR upregulates proteolysis (q = 4.510^-9) and protease-related terms, while downregulated is cell-cycle* [Table: DJ634 - Fatbody/Muscle/Gut].
Upregulated known lifespan-altering genes are Akh, Tequila, CG6048, Ilp2 and Ilp3 as well as Gadd45, while among the downregulated factors are Hsp26, Hsp27, Eip55E, CG5194, ovo, Eip71CD, fs(1)M3 and Hsp68.
Upon DR neurons upregulate immunglobulin-like related terms and downregulate transcripts associated to the nucleus and cell-cycle [Table: ELAV-C155 - Neurons].
Upregulated lifespan-associated genes are Sh, Ilp5, ry, CG4726, Rbp9, tim, bchs, Rh2, Naam, CG6184, SIFR, Hsp68, VhaSFD, NF1, Pdf, Cct1, hebe, bsk, sm, dro4, CG12004, Akh, ImpL2, CG34243, Prm, Hsp23, Ilp2, Ilp3, elav, Scgdelta, dnc, Hk, Hsp70Ba, Hsp70Bc, Hsp70Bb, rut, endoA, Mlp84B and Orco. Downregulated are fabp, kuk, DLP, Trx-2, esc, bam, Pten, magu, Sin3A, POSH, g, Atg7, POSH, insc, pex1, fs(1)M3, kermit, CG13890, CG6511, mth, Hsp26, Hsp27, Bmcp, ovo, Edem1, Doc1 TrxT and p53.
In the heart DR upregulated transcripts are associated to insect cuticle protein, signal peptide, Cytoplasmic, Helix-loop-helix-motif and folate biosynthesis, while it downregulates glycosidase and innate immunity related terms [Table: HAND4.2 - Heart].
Among the known lifespan-controlling genes, Sh, Rdh, CG30427, CG17856, CG13306 and rpr as well as Prm are upregulated, while downregulated are car, Atg7, Eip71CD, Pten, endoA, CG12004, Doc1, fabp, mth, CG18809, g, and kermit.
In the thoracic muscle DR upregulated transcripts are associated with insect cuticle protein, neurotransmitter channels and ion channel/transport, while downregulated are terms related to cell division/cell cycle [Table: MHC - Thoracic Muscle].
Upregulated are Sh, esg, rpr and CG30427, while downregulated are CG5194, Hsp27, Atg7, Bmcp, car, ovo, Pten, mth, Doc1 and kermit.
In the gut tissue transcripts upon DR which are enriched for upregulation are associated to Major facilitator superfamily MFS-1, Trypsin and Peptidase. In contrast, downregulated transcripts are enriched for cell cycle and glutathione S-transferase [Table: Myo1A - Gut].
Upregulated are to (TakeOut), Orco and TrxT, while downregulated is cert, bam, Hsp27, Hsp68, and Pten .
In the germline DR invokes the downregulation of nucleotide binding (q = 1.710^-4), DNA-metabolic processes (q = 4.210^-6) including DNA repair (q = 5.710^-2) as well as regulation of RNA splicing (q = 1.110^-2) and RNA processing (q = 1.110^-5), cell division (q = 1.910^-2). In contrast, DR results in the germline to an upregulates of plasma membrane part (q = 5.610^-7), neurotransmitter receptor activity (q = 5.110^-8) and intrinsic to membrane (q = 2.410^-8) as well as transcription factor activity (q = 1.810^-4), but also protealysis (q = 1.9*10^-3) [Table: NGT - Germline].
Upregulated are Sh, Rdh, Ilp3, TrxT, CG30427 and rpr while downregulated are Pten, car, Gclc, CG12576, foxo, fs(1)M3, pex1, Bmcp, dj-1beta, mth, cher, fh, magu, Doc1, CG16711, Cdk5, cm, hep, Cdk5, endoA, g, CG18809, ovo and kermit.
In the fatbody and gut DR results into the upregulation of ion transport and alternative splicing as well as the downregulation of cell-cycle and Glutathione S-transferase related transcripts [Table: S106 - Fatbody/Gut].
Upregulated are Sh, to (TakeOut), PGRP-LF, VhaSFD, Pdf, Tequila, ImpL2 and InR, while downregulated are the transcripts encoding for numerous heat-shock proteins (Hsp22, Hsp23, Hsp26 and Hsp27) as well as bam, CG5194, CG6041, and kuk.
In head and thorax body compartiments DR significantly upregulates (top 400 genes) proteolysis (q = 2.510^-8), cell division (q = 1.610^-6), including the meiotic cell cycle (q = 5.810^-3) and reproductive cellular process (q = 1.310^-7), maternal tudor protein (q = 4.310^-3), p-granule (q = 8.810^-3) and germ-plasm. The thorax is composed of somatic tissues without sex-tissues [Meiklejohn et al., 2011]. The germ plasm is a differentiated cytoplasm associated with germ cells.
Downregulated by DR in head/thorax are Immunoglobulin-like (q = 4.510^-7), integral to plasma membrane (q = 9.510^-9), gated-channel activity (q = 1.410^-9), neuroactive ligand-receptor interaction (q = 3.110^-7) and transcription factor activity (q = 2.5*10^-4) [Table: HT - Head/Thorax].
Upregulated is bam, esg, esc, Eip71CD, CG5194, CG6041, and fs(1)M3 while downregulated are Sh, rut, Orco, CG10916, CG10383, Gadd45 and Hsp70Bb.
The interactions and communication between different tissues is partly mediated by secretary factor such as hormones. We identified all genes that encode gene products classified be related to the term hormone.
In the Malpighian Tubule many hormone-related genes are upregulated and few were downregulated too.
c42 up
Pdf (pigment-dispersing factor): circadian singalosomes/rhythm, neuropeptide hormone activity, receptor binding
amon (amontillado): peptide hormone processing
Dh44 (Diuretic hormone 44): diuretic/neuropeptide hormone activity
Ilp2 (insulin-like peptide 2): insuling receptor binding, hormone activity
CCHa1 (CCHamide-1): neuropeptide hormone activity
sNPF (short neuropeptide F precursor): neuropeptide hormone activity
capa (capability): diuretic/myostimulatory/neuropeptide hormone acivity, receptor binding
Nplp1 (Neuropeptide-like precursor 1): neuropeptide hormone activity
CG15537: hormone binding
Dsk (Drosulfakinin): neuropeptide hormone activity
Lk (Leucokinin) diuretic/neuropeptide hormone activity
Pk1r (Pyrokinin 1 receptor) G-protein coubpled receptor activity, growth-hormone-releasing homeone receptor activity, neuropeptide receptor activity
Lgr1 G-protein couple receptor activity, neuropeptide receptor activity protein-hormone receptor activity
Proct (Proctolin) neuropeptide hormone activity
GRHR (Gonadotropin-releasing hormone receptor): G-protein coupled receptor activity, adipokinetic hormone binding/receptor activity, gonatropin-releasing hormone receptor activity, neuropeptide receptor activity
Dms (Dromyosuppressin): neuropeptide hormone activity, negative regulation of hear contradiction
Dh31 (Diuretic hormone 31): circadian, diuretic hormone activity, neuropeptide hormone activity
Eip78C (Ecdysone-induced protein 78C): steroid hormone receptor activity, ligand-activated sequence-specific DNA binding RNA polymerase II transcription factor activity
kni (knirps) transcription factor
7B2: neuropeptide signaling pathway, peptide hormone precessing
Ast-C (Allatostatin C): hormone activity, myoinhibitory/neuropeptide hormone activity, negative regulatio of juvenile hormone biosynthetic process
Nplp3 (Neuropeptide-like precursor 3): neuropeptide hormone activity: neuropeptide hormone activity
Ast (Allatostatin): neuropeptide hormone activity
c42 down
burs (bursicon): contribution to neuropeptide hormone activity
Nc (Nedd2-like caspase): protein homodimerization activity
ETH (Ecdysis triggering hormone): ecdysis-triggering hormone activity, neuropeptide hormone activity, receptor binding
In the Fatbody/Muscle/Gut there were only upregulated transcripts related to hormones.
DJ534 up
capa (capability): diuretic/myostimulatory/neuropeptide hormone activity, receptor binding
Akh (Adipokinetic hormone): adipokinetic/neuropeptide hormone activity
Dh31-R1 (Diuretic hormone 31 receptor 1): circadian, calcitonin receptor activity, diuretic/neuropeptide hormone receptor activity
Lk (Leucokinin): diuretic/neuropeptide hormone activity
GRHR (Gonadotropin-releasing hormone receptor): G-protein coupled receptor activity, adipokinetic/neuropeptide hormone receptor activity, gonatropin-releasing hormone receptor activity
Ilp3 (Insulin-like peptide 3): insulin receptor binding, hormone activity
Ast-C (Allatostatin C): myoinhibitory/neuropeptide hormone activity
Ilp2 (Insulin-like peptide 2): insulin receptor binding, hormone activity
CCHa1 (CCHamide-1): neuropeptide hormone activity
Dms (Dromyosuppressin): neuropeptide hormone activity
Jheh3 (Juvenile hormone epoxide hydrolase 3): juvenile hormone catabolic process
In the nervous system even more hormone-related transcripts were upregulated than in the Malpighian Tubule and several were also downregulated.
ELAV-C155 up
Dh44 (Diuretic hormone 44): diuretic/neuropeptide hormone activity
Tk (Tachykinin): neuropeptide hormone activity, receptor binding
Ilp5 (Insulin-like peptide 5): hormone activity, insulin receptor binding
hug (hugin): ecdysis-triggering hormone activity, hormone activity, myostimulatory/neuropeptide hormone activity, neurotrophin receptor binding
svp (seven up): steroid hormone receptor activity, sequence-specific DNA binding
CG8784: G-protein coupled receptor activity, growth hormone-releasing hormone receptor activity, neuropeptide receptor activity
CCHa2 (CCHamide-2): neuropeptide hormone activity
Proct (Proctolin): neuropeptide hormone activity
CCHa1 (CCHamide-1): neuropeptide hormone activity
Crz (Corazonin): neuropeptide hormone activity
npf (neuropeptide F): neuropeptide hormone activity
Eh (Eclosion hormone): ecdysis-triggering/eclosion/neuropeptide hormone activity
Lgr1: G-protein coupled receptor activity, neuropeptide receptor activity, protein-hormone receptor activity
ETHR: G-protein coupled amine receptor activity, ecdysis-triggering hormone receptor activity, neuropeptide receptor activity, neurogenesis
Ccap (Cardioacceleratory peptide): neuropeptide hormone activity
Ast-C (Allatostatin C): myoinhibitory/neuropeptide hormone activity
Ast (Allatostatin): neuropeptide hormone activity
amon (amontillado): peptide hormone processing
Dh31 (Diuretic hormone 31): circadian, diuretic hormone activity, neuropeptide hormone activity
sNPF (short neuropeptide F precursor): neuropeptide hormone activity
capa (capability): diuretic/myostimulatory/neuropeptide hormone activity, receptor binding
dimm (dimmed): positive regulation of hormone secretion, sequence-specific DNA-binding transcription factor activity
CG15537: hormone binding
Fmrf (FMRFamide-related): neuropeptide hormone activity
Hr46 (Hormone receptor-like in 46): thyroid/steroid hormone receptor activity
GRHR (Gonadotropin-releasing hormone receptor): G-protein coupled receptor activity, adipokinetic hormone binding/receptor activity, gonadotropin-releasing hormone receptor activity, neuropeptide receptor activity
Dms (Dromyosuppressin): neuropeptide hormone activity
Dh44-R1 (Diuretic hormone 44 receptor 1): G-protein coupled receptor activity, calcitonin receptor activity, diuretic hormone receptor activity, neuropeptide hormone/receptor activity
Dh31-R1 (Diuretic hormone 31 receptor 1): calcitonin receptor activity, diuretic hormone receptor activity, neuropeptide receptor activity, G-protein coupled receptor singaling pathway, integral to membrane
IFa (IFamide): neuropeptide hormone activity
Hr51 (Hormone receptor 51): circadian rythms, steroid hormone receptor activity, neurogenesis
Eip93F (Ecdysone-induced protein 93F): hormone-mediated apoptotic signaling pathway
Cyp4aa1: hormone metabolic process
Dsk (Drosulfakinin): neuropeptide hormone activity
Nplp1 (Neuropeptide-like precursor 1): neuropeptide hormone activity
itp (ion transport peptide): neuropteptide hormone activity, circadian rhythm
Akh (Adipokinetic hormone): adipokinetic/neuropeptide hormone activity
DOR: steroid hormone receptor binding, ligand-dependent nuclear receptor transcription coactivator activity, regulation of autophagy
Ilp2 (insulin-like peptide 2): insuling receptor binding, hormone activity
Ilp3 (Insulin-like peptide 3): hormone activity, insulin receptor binding
GRHRII: G-protein coupled receptor activity, adipokinetic hormone receptor activity, corazonin receptor activity, gonadotropin-releasing hormone receptor activity, neuropeptide receptor activity, protein-hormone activity
Ilp7 (Insulin-like peptide 7): hormone activity, insulin receptor binding
Eip75B (Ecdysone-induced protein 75B): circadian clock, steroid/thryorid receptor activity
Mip (Myoinhibiting peptide precursor): ecdysiostatic/myoinhibitory/neuropeptide hormone activity
Eip78C (Ecdysone-induced protein 78C): steroid hormone receptor activity, ligand-activated sequence-specific DNA binding RNA polymerase II transcription factor activity
7B2: peptidase activator, neuropetide singaling pathway, peptide hormone processing
Lk (Leucokinin) diuretic/neuropeptide hormone activity
ELAV-C155 down
FK506-bp1 (FK506-binding protein 1): juevenile hormone response element binding
Hr96 (Hormone receptor-like in 96): steroid hormone receptor activity
usp (ultraspiracle): ecdysteroid hormone receptor activity, juvenile hormone binding
ETH (Ecdysis triggering hormone): ecdysis-triggering hormone activity, receptor activity
Nc (Nedd2-like caspase): protein homodimerization activity
jub (Ajuba LIM protein): thyroid hormone receptor binding, ligand-dependent nuclear binding
In the heart several hormone associated transcripts are up and downregulated.
Hand4.2 up
Ilp1 (Insulin-like peptide 1): hormone activity, insulin receptor binding
Lgr1: G-protein coupled receptor activity, neuropeptide receptor activity, protein-hormone receptor activity
Nplp4 (Neuropeptide-like precursor 4): neuropeptide hormone activity
Nplp3 (Neuropeptide-like precursor 3): neuropeptide hormone activity
Nplp1 (Neuropeptide-like precursor 1): neuropeptide hormone activity
Dms (Dromyosuppressin): neuropeptide hormone activity
Cyp4aa1: hormone metabolic process
Ast (Allatostatin): neuropeptide hormone activity
Hand4.2 down
CG8784: G-protein coupled receptor activity, growth hormone-releasing hormone receptor activity, neuropeptide receptor activity
CCHa2 (CCHamide-2): neuropeptide hormone activity
DOR: steroid hormone receptor binding, ligand-dependent nuclear receptor transcription coactivator activity, regulation of autophagy
Jhe (Juvenile hormone esterase): juvenile-hormone esterase activity
In the thoracic muscle several hormones are upregualted, while a pair of others are downregulated.
MHC up
rk (rickets): G-protein coupled receptor activity, neuropeptide receptor activity, protein-hormone receptor activity
Ilp1 Insulin-like peptide 1: hormone activity, insulin receptor binding
Nplp1 (Neuropeptide-like precursor 1): neuropeptide hormone activity
Dh44-R2 (Diuretic hormone 44 receptor 2): G-protein coupled receptor activity, calcitonin receptor activity, diuretic hormone receptor activity, neuropeptide hormone acitivty, neuropeptide receptor activity
burs (bursicon): contributes to neuropeptide hormone activity
Ast-C (Allatostatin C): myoinhibitory/neuropeptide hormone activity
MHC down
Nc (Nedd2-like caspase): induction of programmed cell death by hormone
Ilp7 (Insulin-like peptide 7): hormone activity, insulin receptor binding
In the Gut one hormone capa is upregulated and 4 other hormone related transcripts are downregulated.
Myo1A up
Myo1A down
Nc (Nedd2-like caspase): induction of programmed cell death by hormones
CCHa2 (CCHamide-2): neuropeptide hormone activity
ETH (Ecdysis triggering hormone): receptor binding
Hr96 (Hormone receptor-like in 96): steroid hormone receptor binding, sequence-specific DNA binding transcription factor activity
In the germline a considerable amount of hormones and receptors are being upregulated and one hormone is downregulated, CCHa2.
NGT up
Ilp1 (Insulin-like peptide 1): hormone activity, insulin receptor binding
Ilp3 (Insulin-like peptide 3): hormone activity, insulin receptor binding
Jheh3 (Juvenile hormone epoxide hydrolase 3): juvenile hormone catabolic process
Nplp4 (Neuropeptide-like precursor 4): neuropeptide hormone activity
Nplp3 (Neuropeptide-like precursor 3): neuropeptide hormone activity
Nplp1 (Neuropeptide-like precursor 1): neuropeptide hormone activity
Eh (Eclosion hormone): ecdysis-triggering/eclosion/neuropeptide hormone activity
GRHR (Gonadotropin-releasing hormone receptor): G-protein coupled receptor activity, adipokinetic hormone binding/receptor activity, gonadotropin-releasing hormone receptor activity, neuropeptide receptor activity
ETHR: G-protein coupled amine receptor activity, ecdysis-triggering hormone receptor activity, neuropeptide receptor activity, neurogenesis
Dh44-R1 (Diuretic hormone 44 receptor 1): G-protein coupled receptor activity, calcitonin receptor activity, diuretic hormone receptor activit, neuropeptide hormone/receptor activity
NGT down
In the fatbody/gut numerous hormone associated transcripts are upregualted and one is downregulated.
S106 up
Pdf (Pigment-dispersing factor): neuropeptide hormone activity, receptor binding
itp (ion transport peptide): neuropteptide hormone activity, circadian rhythm
Ilp6 (Insulin-like peptide 6): hormone binding, insulin receptor binding
svp (seven up): steroid hormone receptor activity, sequence-specific DNA binding
Nplp1 (Neuropeptide-like precursor 1): neuropeptide hormone activity
Proct (Proctolin): neuropeptide hormone activity
GRHR (Gonadotropin-releasing hormone receptor): G-protein coupled receptor activity, adipokinetic hormone binding/receptor activity, gonatropin-releasing hormone receptor activity, neuropeptide receptor activity
Cyp4aa1: hormone metabolic process
Ast (Allatostatin): neuropeptide hormone activity
7B2: peptidase activator, neuropetide singaling pathway, peptide hormone processing
Ast (Allatostatin): neuropeptide hormone activity
Nplp3 (Neuropeptide-like precursor 3): neuropeptide hormone activity
S106 down
The most significant interactors of the differential, up and down-regulated genes of the respective signatures were determined [Table: Network Enrichment]. Notable micro-RNAs are frequently among the candidates of the significant interactors.
The functional enrichment of the significant interactors (p-value
From the results here it appears that DR commonly upregulates RNA-editing and alternative splicing as well as downregulates juvenile hormone signalling.
Overall in the majority of tissues immuglobulin-like protein encoding transcripts were also enriched for upregulation (q
Surprisingly the gene expression changes of the first eight tissues are exactly the opposite of the changes previously reported in the head-thorax [Antosh et al., 2011]. Statistical analysis confirms that genes upregulated in head-thorax are not the same as those reported here, neither the downregulated. However, those genes upregulated in the eight other tissues are significantly enriched for downregulation in the head-thorax signature and vice versa. The most likely explanation for this discrepancy is that the labels (DR and AL) either in the previous study or in this study were accidentally exchanged.
The lifespan extension by DR is mediated by specific genetic pathways. Several factors including specific genes and metabolites were shown to mediate the effect of nutrient-sensing signalling on lifespan. Manipulation of defined factors can greatly alter the lifespan extension response to DR or even totally abolish it and such genes are therefore said to be essential for DR (DR-essential genes) [22912585].
The insulin like peptides (ILPs) Ilp2, Ilp3 and Ilp5, if mutated together reverse the DR lifespan extension effect. Ilp2 is transcriptional downregulated in long-lived mutants and its mutation extends the lifespan. Ilp5 mRNA level was previously also found to decline under DR [20195512]. However, here we found that ILPs are transcriptional upregulated in numerous tissues. For instance, Ilp5 is upregulated in the ELVAC-C155 (neurons), Ilp2 is upregulated in the c42 (malpighian tubule), DJ634 (fatbody/muscle/gut) and ELAV-C155, while Ilp3 is upregulated in the ELAV-C155 and DJ634, as well as in the NGT (germline).
Similar the single insulin-like receptor encoded in the genome of Drosophila was found to be upregulated in S106 (fatbody/gut).
The functional ortholog of glucagon, the adipokinetic hormone (Akh), was upregulated in DJ634 (fatbody/muscle/gut) and ELAV-C155 (neurons). Ubiquitinous or fatbody specific over-expression of Akh was shown to extend the lifespan in an non-additive manner with DR.
The transcript encoding the juvenile hormone binding protein TakeOut is upregulated in Myo1A (gut) and S106 (fatbody). Previously it was found that starvation, DR and numerous longevity mutants upregulate TakeOut and its over-expression in either neurons or fatbody was sufficient to extend the lifespan.
Loss of function of the odorant receptor Orco extends the lifespan. Orco mutant's lifespan is further extended by DR, but to a significant lesser degree than on AL. Orco is upregulated in c42 (malpighian tubule), ELAV-C155 (neurons), Myo1A (gut), but downregulated in head/thorax.
The transcript encoding reaper (rpr) is upregulated in c42 (malpighian tubule) and MHC (thoracic muscle). Overexpression of reaper reduces the lifespan extension conferred by DR.
Manipulation of the activity of p53 is required for the DR-lifespan extension response. A dominant negative form of p53 specifically expressed in neurons extends the lifespan and cancels out the lifespan extension by DR. Here we found that DR downregulates the transcript level of p53 exactly in this tissue.
The forkhead transcription factors were often found to be very central in the response of altered nutrient sensing on lifespan, although their role in DR is controversial.
foxo is slightly downregulated by DR in the germline and gut, thoracic muscle, malpighian tubule, neurons and heart, while both slightly downregulated and slightly upregulated in the S106 (fatbody/gut) and (DJ634) (fatbody/muscle/gut).
foxo is a nutrient-responsive transcription factor which is often found to be upregulated in lifespan-extending interventions (particularly by suppression of insulin-like signalling). However, transcripts encoded by the foxo gene are in the majority of tissues slightly downregulated, rather than upregulated, except in two cases where a single isoform was also enriched for slight upregulation. Upregulation of foxo in the fatbody is totally sufficient to extend the lifespan [18241326].
Inhibition of the gamma-cystationinase encoded by Eip55E via propargylglycine treatment suppressed DR lifespan prolongation. Eip55E is downregulated by DR in the DJ634 (fatbody/muscel/gut) and ELAV-C155 (neurons).
Overall DR triggers the upregulated of many more hormone-related transcripts than it downregulates.
Female animals were kept on a DR diet (0.5% yeast extract) or ad libitum (5% yeast extract). Both diets contain 1.8% sugar, some agar and corn meal. The flies were sorted on food 14 days setting up the cross, then kept on the corresponding food for 12 days (change out every other day). Killing before gene expression profiling was performed with CO2 and tissue harvested around 12 to 2 pm.
Molecular profiles were grouped according to body-compartment/tissue [Table: Tissues utilized to investigate the DR effect], gene expression intensities averaged among replicates (5-6) within a group and condition (i.e. DR and AL) and ratio between experimental (DR) and control group (AL) calculated. Those genes which were more than two-fold upregulated and reached a significance below 0.05 were deemed as differential expressed, if nothing else were specified. Benjamini Hochberg correction was applied in most cases and those p-values designated as q-values. Whether two transcripts/gene sets overlap more as expected by chance were evaluated with the hypergeometric test.
Interaction networks were created and analysed as previously described.
Antosh et al., 2011: Antosh M, Whitaker R, Kroll A, Hosier S, Chang C, Bauer J, Cooper L, Neretti N, Helfand SL (2011) Comparative transcriptional pathway bioinformatic analysis of dietary restriction, Sir2, p53 and resveratrol life span extension in Drosophila. Cell cycle (Georgetown, Tex.) 10: 904-11.
Meiklejohn et al., 2011: Meiklejohn CD, Landeen EL, Cook JM, Kingan SB, Presgraves DC (2011) Sex chromosome-specific regulation in the Drosophila male germline but little evidence for chromosomal dosage compensation or meiotic inactivation. PLoS biology 9: e1001126.
Giannakou et al., 2008: Giannakou ME, Goss M, Partridge L (2008) Role of dFOXO in lifespan extension by dietary restriction in Drosophila melanogaster: not required, but its activity modulates the response. Aging cell 7: 187-98.
Table: Tissues utilized to investigate the DR effect.
Signature Tissue Up Down
c42 malpighian tubule 1136 827
DJ634 fatbody, but also muscle and gut 238 466
ELAV-C155 neurons 2825 2502
HAND4.2 heart 1361 839
MHC thoracic muscle 405 973
Myo1A gut 327 326
NGT germline 594 581
S106 fatbody, but also gut 672 754
Table: Effects of DR common to multiple tissues 1.
Term q-value Direction
rna editing 2.2*10^-5 Up
synapse 1.9*10^-4 Up
membrane 1.8*10^-4 Up
cell division 4.4*10^-12 Down
cell cycle 4.1*10^-11 Down
mitosis 6.8*10^-8 Down
Table: Effects of DR common to multiple tissues 2.
Term q-value Direction
vision 0.02 Up
alternative splicing 0.02 Up
sensory transduction 0.03 Up
IPR005649:Chorion 2 0.01 Down
Secreted 0.01 Down
IPR004272:Odorant binding protein 0.03 Down
SM00700:JHBP 0.02 Down
IPR013053:Hormone binding 0.03 Down
Table: c42 - Malpighian Tubule.
Term q-value Direction
IPR007110:Immunoglobulin-like 6.51*10^-9 Up
IPR003598:Immunoglobulin subtype 2 8.11*10^-9 Up
signal peptide 7.71*10^-8 Up
cell cycle 2.61*10^-16 Down
mitosis 1.31*10^-15 Down
cell division 1.41*10^-15 Down
Table: DJ634 - Fatbody/Muscle/Gut.
Term q-value Direction
SM00020:Tryp_SPc 1.8*10^-10 Up
IPR001314:Peptidase S1A, chymotrypsin 2.6*10^-9 Up
IPR018114:Peptidase S1/S6, chymotrypsin/Hap, active site 1.8*10^-9 Up
dna replication 2.1*10^-11 Down
cell division 1.7*10^-11 Down
cell cycle 5.1*10^-11 Down
Table: ELAV-C155 - Neurons.
Term q-value Direction
IPR007110:Immunoglobulin-like 7.8e-37 Up
receptor 3.0e-36 Up
transmembrane 2.2-36 Up
nucleus 1.6e-15 Down
cell cycle 1.8e-14 Down
dme03030:DNA replication 2.2e-14 Down
Table: HAND4.2 - Heart.
Term q-value Direction
IPR000618:Insect cuticle protein 3.0e-11 Up
IPR002557:Chitin binding protein, peritrophin-A 1.7e-05 Up
SM00494:ChtBD2 4.0e-05 Up
glycosidase 1.1e-05 Down
IPR013781:Glycoside hydrolase, subgroup, catalytic core 0.00037 Down
signal peptide 0.00056 Down
Table: MHC - Thoracic Muscle.
Term q-value Direction
PR006029:Neurotransmitter-gated ion-channel transmembrane region 4.0e-06 Up
PIRSF002443:acetylcholine receptor 9.5e-07 Up
IPR006201:Neurotransmitter-gated ion-channel 6.1e-06 Up
cell division 2.2e-12 Down
mitosis 8.5e-12 Down
cell cycle 3.4e-11 Down
Table: Myo1A - Gut.
Term q-value Direction
IPR007484:Peptidase M28 0.0075 Up
IPR011701:Major facilitator superfamily MFS-1 0.0080 Up
PIRSF001135:trypsin 0.00132502439997 0.0049 Up
IPR013135:Vitelline membrane cysteine-rich domain 1.0e-05 Down
SM00703:NRF 4.3e-05 Down
IPR002656:Acyltransferase 3 0.00012 Down
Table: NGT - Germline.
Term q-value Direction
ionic channel 6.4e-05 Up
synapse 8.1e-05 Up
splice variant 0.00036 Up
phosphoprotein 0.00063 Down
helicase 0.00088 Down
atp-binding 0.0016 Down
Table: S106 - Fatbody/Gut.
Term q-value Direction
ion transport 2.1e-09 Up
ionic channel 1.3e-08 Up
splice variant 1.1e-06 Up
dna replication 5.0e-06 Down
IPR004045:Glutathione S-transferase, N-terminal 3.1e-05 Down
IPR004046:Glutathione S-transferase, C-terminal 3.1e-05 Down
Table: HT - Head/Thorax.
Term q-value Direction
mitosis 5.2e-12 Up
cell division 1.8e-11 Up
IPR001314:Peptidase S1A, chymotrypsin 1.3e-09 Up
IPR013783:Immunoglobulin-like fold 3.2e-05 Down
IPR003598:Immunoglobulin subtype 2 6.6e-05 Down
IPR007110:Immunoglobulin-like 4.8e-05 Down
Network Candidates.
c42 Up mir-4 mir-1000 mir-1002
Delta su(HW) chif rec
Down mip130 E2f2 chif
DJ634 Up su(Hw) mir-927 mir-263b
Delta chif DNApol-alpha50 rec
Down chif rec DNApol-alpha50
ELAV-C155 Up mir-1011 mir-137 mir-316
Delta Myb Trl sens
Down Myb lin-52 mip130
Hand4.2 Up su(Hw) chinmo mir-1014
Delta mir-1011 mir-33 mir-4
Down chinmo Aats-gly CG15211
MHC Up mir-33 mir-1002 mir-991
Delta Cp190 Chro BEAF-32
Myo1A Up su(Hw) Mlc2 Adh
Delta su(Hw) E2f2 chinmo
Down E2f2 sens mip130
NGT Up mir-316 mir-iab-4 mir-33
Delta CG6695 CG6796 Jon74E
Down Myb mip120 BEAF-32
S106 Up mir-1000 mir-315 su(Hw)
Delta su(Hw) mir-8 Trl
Down chif E2f2 DNApol-alpha50
c42 delta [http://denigma.de//admin/network/candidate/?query=c42+Delta&taxid=7227&o=7] c42 up [http://denigma.de/admin/network/candidate/?query=c42+Up&taxid=7227&o=7] c42 down [http://denigma.de/admin/network/candidate/?query=c42+Down&taxid=7227&o=7] DJ634 delta [http://denigma.de/admin/network/candidate/?query=DJ634+Delta&taxid=7227&o=7] DJ634 up [http://denigma.de/admin/network/candidate/?query=DJ634+up&taxid=7227&o=7] DJ634 down [http://denigma.de/admin/network/candidate/?query=DJ634+Down&o=7] Hand4.2 delta [http://denigma.de/admin/network/candidate/?query=Hand4.2+Delta&taxid=7227&o=7] Hand4.2 up [http://denigma.de/admin/network/candidate/?query=Hand4.2+Up&taxid=7227&o=7] Hand4.2 down [http://denigma.de/admin/network/candidate/?query=Hand4.2+Down&taxid=7227&o=7] MHC delta [http://denigma.de/admin/network/candidate/?query=MHC+Delta&taxid=7227&o=7] MHC up [http://denigma.de/admin/network/candidate/?query=MHC+Up&taxid=7227&o=7] Myo1A up [http://denigma.de/admin/network/candidate/?query=Myo1A+Up&taxid=7227&o=7] Myo1A delta [http://denigma.de/admin/network/candidate/?query=Myo1A+Delta&taxid=7227&o=7] Myo1A down [http://denigma.de/admin/network/candidate/?query=Myo1A+Down&taxid=7227&o=7] NGT delta [http://denigma.de/admin/network/candidate/?query=NGT+Delta&taxid=7227&o=7] NGT up [http://denigma.de/admin/network/candidate/?query=NGT+Up&taxid=7227&o=7] S106 Delta [http://denigma.de/admin/network/candidate/?query=S106+Delta&taxid=7227&o=7] S106 up [http://denigma.de/admin/network/candidate/?query=S106+Up&taxid=7227&o=7] S106 down [http://denigma.de/admin/network/candidate/?query=S106+Down&taxid=7227&o=7]
System Message: ERROR/3 (, line 758)
Error parsing content block for the "table" directive: exactly one table expected.
.. table:: Table: Network Enrichment.
+---------+------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
|Tissue |Effect|Term |T |S |R |p-value |
+---------+------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
|c42 |Up |electron carrier activity |334 |152|0.46|4.22e-79|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |microsome |85 |50 |0.59|3.03e-34|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen|89 |49 |0.55|4.39e-32|
| +------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| |Down |DNA replication |66 |47 |0.71|1.71e-40|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |nuclear origin of replication recognition complex |18 |18 |1.0 |3.47e-22|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |DNA-dependent DNA replication initiation |16 |15 |0.94|1.16e-18|
+---------+------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
|DJ634 |Up |electron carrier activity |334 |65 |0.19|1.77e-31|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |microsome |85 |21 |0.25|1.78e-13|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen|89 |20 |0.22|3.37e-12|
| +------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| |Down |DNA replication |66 |40 |0.61|2.66e-37|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |DNA-dependent DNA replication initiation |16 |14 |0.88|2.10e-19|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |nuclear origin of replication recognition complex |18 |15 |0.83|1.90e-18|
+---------+------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
|ELAV-C155|Up |electron carrier activity |334 |158|0.47|1.60e-65|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen|89 |51 |0.57|6.08e-28|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |microsome |85 |49 |0.58|8.54e-27|
| +------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| |Down |nucleus |1579|277|0.18|0.0 |
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |nuclear mRNA splicing, via spliceosome |301 |89 |0.30|2.23e-30|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |DNA replication |66 |31 |0.47|4.53e-17|
+---------+------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
|Hand4.2 |Up |electron carrier activity |334 |62 |0.19|1.14e-14|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |proton transport |33 |16 |0.49|1.82e-11|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |hydrogen-exporting ATPase activity, phosphorylative mechanism |62 |22 |0.35|5.31e-10|
| +------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| |Down |electron carrier activity |334 |112|0.34|1.46e-76|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |microsome |85 |37 |0.44|5.85e-32|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen|89 |36 |0.40|2.69e-30|
+---------+------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
|MHC |Up |acetylcholine-activated cation-selective channel activity |18 |12 |0.67|1.02e-12|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |acetylcholine-gated channel complex |16 |11 |0.69|4.68e-12|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |compartment pattern specification |6 |5 |0.83|4.51e-07 |
| +------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| |Down |spindle |36 |14 |0.39|2.63e-10|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |DNA replication |66 |18 |0.27|5.51e-10|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |nuclear origin of replication recognition complex |18 |10 |0.56|1.20e-09 |
+---------+------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
|Myo1A |Up |electron carrier activity |334 |142|0.43|4.17e-95|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |microsome |85 |46 |0.54|4.56e-38|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen|89 |43 |0.48|2.03e-33|
| +------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| |Down |glutathione transferase activity |57 |26 |0.46|5.61e-19|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |kinetochore |38 |20 |0.53|1.20e-15|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |mitotic cell cycle spindle assembly checkpoint |18 |14 |0.78|2.34e-15|
+---------+------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
|NGT |Up |electron carrier activity |334 |159|0.48|3.25e-89|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |microsome |85 |53 |0.62|2.26e-39|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen|89 |52 |0.58|4.80e-37|
| +------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| |Down |nuclear mRNA splicing, via spliceosome |301 |64 |0.21|1.77e-25|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |spliceosomal complex |69 |24 |0.35|3.43e-15|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |recatalytic spliceosome |141 |27 |0.19|2.20e-10|
+---------+------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
|S106 |Up |electron carrier activity |334 |147|0.44|4.22e-80|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen|89 |47 |0.53|4.86e-32|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |microsome |85 |46 |0.54|1.06e-31|
| +------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| |Down |electron carrier activity |334 |135|0.40|3.30e-84|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |microsome |85 |45 |0.53|2.64e-36|
| | +-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
| | |oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen|89 |44 |0.49|2.21e-34|
+---------+------+-----------------------------------------------------------------------------------------------------+----+---+----+-------------------+
c42 up [http://denigma.de/admin/network/enrichment/?analysis__id__exact=74&o=5] c42 down [http://denigma.de/admin/network/enrichment/?analysis__id__exact=76&o=5] DJ634 up [http://denigma.de/admin/network/enrichment/?analysis__id__exact=78&o=5] DJ634 down [http://denigma.de/admin/network/enrichment/?analysis__id__exact=79&o=5] ELAV-C155 up [http://localhost:7777/admin/network/enrichment/?analysis__id__exact=81&o=5] ELAV-C155 Down [http://denigma.de/admin/network/enrichment/?analysis__id__exact=82&o=5] Hand4.2 Up [http://denigma.de/admin/network/enrichment/?analysis__id__exact=84&o=5] Hand4.2 Down [http://denigma.de/admin/network/enrichment/?analysis__id__exact=85&o=5] MHC Up [http://denigma.de/admin/network/enrichment/?analysis__id__exact=87&o=5] MHC down [http://denigma.de/admin/network/enrichment/?analysis__id__exact=88&o=5] Myo1A up [http://denigma.de/admin/network/enrichment/?analysis__id__exact=90&o=5] Myo1A down [http://denigma.de/admin/network/enrichment/?analysis__id__exact=91&o=5] NGT up [http://denigma.de/admin/network/enrichment/?analysis__id__exact=93&o=5] NGT down [http://denigma.de/admin/network/enrichment/?analysis__id__exact=94&o=5] S106 up [http://denigma.de/admin/network/enrichment/?analysis__id__exact=96&o=5] S106 down [http://denigma.de/admin/network/enrichment/?analysis__id__exact=97&o=5]