Abstract:The interallations between the circadian clock and dietary restriction mediated lifespan extension are unclear. Circadian rhythmicity decreases with age and dietary restriction enhances the amplitude of circadian oscillations to youthful levels. Whether the circadian clock has a causal role in the lifespan extension by a restricted diet remains unknown. Here we analysed the gene expression differences around the circadian cycle under dietary restriction in wild-type and timeless mutant with impaired circadian rhythmicity. We found that dietary restriction robustly upregulates Shaker and takeout.
During aging there is a concomitant decline in circadian rhythms and the overal homeostasis of the organism. In flies and mammals (including humans) robust high-amplitude circadian rhythms are present in young individuals but lose their strength wit age [22217096; 22395806; 21454295].
In both mammals and flies aging is associated with reduced expression of specific clock genes in peripheral clocks, but not in the central pacemaker neurons or suprachiasmatic cell nucleus [22217096; 12149444; 22268765].
Clock proteins in Drosophila are expressed in the fat body, an organ that combines the fat and liver functions of mammals.
In mammals there are daily circadian oscillations in hormones such as leptin, insulin, and glucagon, which affect blood glycemia [22705307; 22672634; 19854863].
Diapause expression extends lifespan and delays sensecence [68707322]. Variation in diapause have also be linked to variation at both timeless (tim) a light dependent component of the circadian clock and and the insulin-regulated phosphatidylinositol 3-kinase (PI3-kinase) gene Dp110 (age-1 homolog) [17043223].
tim is a major clock component in circadian rhythms. tim might be an aging gene [19182541].
In this study here we defined the two Classes Lifespan Factors and Circadian Factors for Factors that control the life and daily clock, respectively, and checked for their overlap in wild-type and the timeless mutant.
In the following are the significant deferentially expressed Lifespan and Circadian Factors upon Dietary Restriction in wild-type and timeless mutant strains.
up Sh, to, Prm, elav, CG6048 and Rh2.
down Pten
up: Nplp1, Nplp3, Dh31 and DOR.
down: Lgr3, ETH, Ilp7
up: synapse, ion transport and cell junction
down: signal peptide, Secreted and innate immunity
up: Sh, to, Rdh, PGRP-LF, Prm, Tequila, LanB2, elav, Rbp9, tim, CG6048, CG30427, CG13306, hebe, Rh2, ImpL2 and Mlp84B.
down: Hsp68
up: disulfide bond, splice variant, muscle protein.
down: signal peptide, secreted, and glycosidase
up: Sh, to, Tequila, PDF, elav, Rbp9, CG6048, CG13306, Rh2, Mlp84B
down: Pten
up: ion transport, synapse and ionic channel
down: glycosidase, hydrolase, signal and peptide
up: Sh, to, Prm, Rbp9, CG6048, CG13306, Rh2, Mlp84B
down: Eip71CD, Doc1, Pten
up: ion transport, synapse, vision
down: glycosidase, signal peptide, secreted
up: ion transport, synapse, ionic channel
down: signal peptide, glycosidase, storage protein
up: Sh, to, Prm, Tequila, Scgdelta, Rbp9, CG6048, CG30427, Rh2, Mlp84B
down: dro2, Hsp68, Pten
up: ion transport, synapse, IPR007110:Immunoglobulin-like
down: signal peptide, Secreted, glycosidase
up: Sh, to, Or, Tequila, PGRFP-LF, elav, Rbp9, tim, CG6048, CG13306, Rh2, Mlp84B
down: Hsp23, ovo, Ei71CD, Doc1
up: Sh, to, Prm, Scgdelta, Rbp9, tim, CG6048, Rh2, Mlp84B
down: Ilp5, Hsp23
up: Sh, to, Prm, Rbp9, tim, CG6048, Rh2
down: Hsp23
up: Sh, to, Prm, Tequila, elav, Scgdelta, Rbp9, tim, CG6048, CG133-6, hebe, CG6184, Rh2, Mlp84B
down: Hsp23, ovo, Eip71CD, Doc1, Pten
up: Sh, to, Prm, elav, Rbp9, tim, CG6048, CG13306, Rh2, Mlp84B
down: Eip71CD, Hsp23
up: Sh, to, Prm, Rbp9, tim, CG6048, CG13306, Rh2
down: Hsp23, dro2, Eip71CD
up: Sh, to, Rdl, tim, qvr, slo
down: nocte
[http://localhost:7777/admin/network/enrichment/?analysis__id__exact=99&o=5]:
Z disc 21 12 0.571428571429 6.58345587211e-10 proton transport 33 14 0.424242424242 1.89402209353e-09 mesoderm development 79 21 0.26582278481 1.19622187031e-08
[http://localhost:7777/admin/network/enrichment/?analysis__id__exact=100&o=5]:
electron carrier activity 334 87 0.260479041916 2.73784570096e-58 glutathione transferase activity 57 25 0.438596491228 2.38074302246e-25 microsome 85 28 0.329411764706 2.94722514497e-23
[http://localhost:7777/admin/network/enrichment/?analysis__id__exact=102&o=5]:
electron carrier activity 334 91 0.27245508982 1.45509403165e-27 high affinity inorganic phosphate:sodium symporter activity 20 19 0.95 1.67577565683e-20 transmembrane transport 289 72 0.249134948097 1.04482572534e-16
[http://localhost:7777/admin/network/enrichment/?analysis__id__exact=103&o=5]:
electron carrier activity 334 94 0.281437125749 5.13953794331e-63 microsome 85 31 0.364705882353 3.87739388194e-26 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen 89 30 0.337078651685 1.30746842785e-24
[http://localhost:7777/admin/network/enrichment/?analysis__id__exact=105&o=5]:
electron carrier activity 334 88 0.263473053892 9.78769324706e-26 high affinity inorganic phosphate:sodium symporter activity 20 17 0.85 8.74121531332e-17 acetylcholine-activated cation-selective channel activity 18 15 0.833333333333 3.62080385465e-13
[http://localhost:7777/admin/network/enrichment/?analysis__id__exact=106&o=5]:
electron carrier activity 334 123 0.368263473054 1.41913354606e-96 microsome 85 42 0.494117647059 2.24953021387e-41 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen 89 40 0.449438202247 3.98942375528e-38
[http://localhost:7777/admin/network/enrichment/?analysis__id__exact=108&o=5]:
G-protein coupled receptor signaling pathway 194 38 0.19587628866 4.03197263344e-10 plasma membrane 474 67 0.14135021097 2.53098941745e-09 aminoacylase activity 11 9 0.818181818182 3.3269293946e-09
[http://localhost:7777/admin/network/enrichment/?analysis__id__exact=109&o=5]:
electron carrier activity 334 70 0.209580838323 1.27306129111e-40 microsome 85 23 0.270588235294 4.66467277009e-17 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen 89 23 0.258426966292 7.36419151041e-17
[http://localhost:7777/admin/network/enrichment/?analysis__id__exact=111&o=5]:
acetylcholine-activated cation-selective channel activity 18 12 0.666666666667 3.00168650091e-09 mesoderm development 79 23 0.291139240506 7.99232214254e-09 acetylcholine-gated channel complex 16 11 0.6875 8.66126264369e-09
[http://localhost:7777/admin/network/enrichment/?analysis__id__exact=112&o=5]:
electron carrier activity 334 129 0.38622754491 1.25139763745e-107 microsome 85 43 0.505882352941 5.84066909153e-44 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen 89 42 0.47191011236 2.72227042875e-42
[http://localhost:7777/admin/network/enrichment/?analysis__id__exact=114&o=5]:
electron carrier activity 334 143 0.428143712575 3.67249368346e-76 microsome 85 47 0.552941176471 1.58832450144e-32 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen 89 45 0.505617977528 3.21597480596e-29
[http://localhost:7777/admin/network/enrichment/?analysis__id__exact=115&o=5]:
gamma-tubulin ring complex 10 6 0.6 5.73040147129e-08 lipid metabolic process 92 14 0.152173913043 1.69360964403e-07 negative regulation of transcription from RNA polymerase II promoter 66 12 0.181818181818 1.86309103976e-07
Comparing the effect of the timeless mutation on the gene expression activity changes under dietary restriction reveals that DR independent of the presence of the timeless gene upregulates Sh, to, Pr and CG6048 as known lifespan factors. However the timeless mutant downregulates the aging-suppressor Hsp23, while the wild-type does not. The tim-specific upregulated genes that are not upregulated in wild-type by DR are involved in alternative splicing and EGF as well as GPCR, rhodopsin-like superfamily. In contrast transcripts upregulated only in wild-type but not in timeless mutant are associated with ion transport and monovalent inorganic cation transport. 16 transcripts, with no obvious functional enrichment, were only significantly down-regulated in wild-type but not timeless mutant. Interestingly a greater amount of transcripts are only downregulated in timeless upon DR but not wild-type. Those 84 transcripts are enriched for terms relate to chorion (q-value = 1.7E-17) and external encapsulating structure (q-value = 1.6E-17) as well as Peptidase A1 (q-value = 5.0E-2).
By a 1.5 fold change cut-off cell division and mitosis are significantly downregulated in timeless mutants upon DR if compared with wild type under DR.
SP_PIR_KEYWORDS cell division 11 7.33333333333 1.3552235761e-09 145 15.8820912125 1.09773105605e-07 1.09773105605e-07 1.44430600812e-06 SP_PIR_KEYWORDS mitosis 9 6.0 8.03536362995e-08 145 15.7971602434 6.50862362062e-06 3.25431710557e-06 8.56354595458e-05
The effect of DR on Timeless compared to wild-type on the interaction network level are given below in terms of functional enrichment (only the top three terms are listed.
[http://denigma.de/admin/network/enrichment/?analysis__id__exact=135&o=5]:
electron carrier activity 334 118 0.353293413174 5.34369664923e-117 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen 89 39 0.438202247191 1.01719255189e-46 microsome 85 39 0.458823529412 3.20697553343e-46
[http://denigma.de/admin/network/enrichment/?analysis__id__exact=135&o=5]:
neuron cell-cell adhesion 3 3 1.0 1.75397258568e-07 chitinase activity 29 5 0.172413793103 6.68115576503e-07 chromatin insulator sequence binding 6 3 0.5 5.58192894298e-06
[http://denigma.de/admin/network/enrichment/?analysis__id__exact=138&o=5]:
electron carrier activity 334 90 0.269461077844 1.95694637854e-67 microsome 85 31 0.364705882353 6.6962116258e-29 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen 89 30 0.337078651685 2.28751382502e-27
[http://denigma.de/admin/network/enrichment/?analysis__id__exact=139&o=5]:
ribonucleoprotein complex 28 12 0.428571428571 1.37788758012e-12 G-protein coupled photoreceptor activity 22 9 0.409090909091 3.63712235421e-10 G-protein coupled receptor kinase activity 8 6 0.75 7.81988043066e-10
[http://denigma.de/admin/network/enrichment/?analysis__id__exact=141&o=5]:
integral to membrane 1177 107 0.0909090909091 0.0 transmembrane transport 289 61 0.21107266436 7.53613274109e-38 high affinity inorganic phosphate:sodium symporter activity 20 20 1.0 7.48636271105e-34
[http://denigma.de/admin/network/enrichment/?analysis__id__exact=142&o=5]:
chitinase activity 29 5 0.172413793103 3.48814013615e-06 chromatin insulator sequence binding 6 3 0.5 4.54280645486e-06 aromatic-L-amino-acid decarboxylase activity 6 3 0.5 7.79083548182e-06
[http://denigma.de/admin/network/enrichment/?analysis__id__exact=144&o=5]:
integral to membrane 1177 110 0.0934579439252 0.0 electron carrier activity 334 119 0.35628742515 4.91737646346e-102 microsome 85 41 0.482352941176 2.91609873445e-43
[http://denigma.de/admin/network/enrichment/?analysis__id__exact=145&o=5]:
negative regulation of transcription from RNA polymerase II promoter 66 13 0.19696969697 2.10763660633e-11 G-protein coupled receptor kinase activity 8 6 0.75 2.10214542837e-10 serine-type endopeptidase inhibitor activity 74 11 0.148648648649 1.37886414213e-08
[http://denigma.de/admin/network/enrichment/?analysis__id__exact=147&o=5]:
electron carrier activity 334 44 0.131736526946 4.20470664553e-27 triglyceride lipase activity 40 18 0.45 7.24309160497e-22 lipid metabolic process 92 22 0.239130434783 8.9233749822e-20
[http://denigma.de/admin/network/enrichment/?analysis__id__exact=148&o=5]:
serine-type endopeptidase inhibitor activity 74 13 0.175675675676 4.10648656322e-12 G-protein coupled photoreceptor activity 22 9 0.409090909091 1.31830639983e-11 G-protein coupled receptor kinase activity 8 6 0.75 2.58698345285e-11
[http://denigma.de/admin/network/enrichment/?analysis__id__exact=150&o=5]:
lipid metabolic process 92 30 0.326086956522 2.10833910642e-27 triglyceride lipase activity 40 19 0.475 4.68861111061e-21 electron carrier activity 334 39 0.116766467066 2.30683288477e-19
[http://denigma.de/admin/network/enrichment/?analysis__id__exact=151&o=5]:
alkaline phosphatase activity 16 13 0.8125 4.82909854682e-21 eclosion rhythm 18 9 0.5 3.9495881768e-12 locomotor rhythm 42 11 0.261904761905 9.51069241324e-11
mir-1000 82 1752 0.046803652968 0.0 mir-315 102 2513 0.0405889375249 0.0 su(Hw) 91 1487 0.0611970410222 0.0
CG17658 3 3 1.0 4.67102985812e-07 sens 81 6318 0.0128205128205 7.62875143695e-06 mir-1014 32 1890 0.0169312169312 1.66563805991e-05
mir-1000 185 1752 0.105593607306 0.0 mir-1006 160 1651 0.0969109630527 0.0 mir-1014 174 1890 0.0920634920635 0.0
chinmo 31 2123 0.0146019783325 1.26124271582e-08 sens 68 6318 0.0107628996518 9.51700650509e-08 mir-991 26 1700 0.0152941176471 5.37738808093e-07
mir-1014 124 1890 0.0656084656085 0.0 mir-985 147 2484 0.0591787439614 0.0 mir-283 99 1377 0.0718954248366 0.0
mir-263b 15 1108 0.0135379061372 9.5635629217e-06 MSBP 5 73 0.0684931506849 3.23785399697e-05 Cyp4g1 6 122 0.0491803278689 3.53024784991e-05
mir-4 79 1267 0.0623520126283 0.0 mir-1000 105 1752 0.0599315068493 0.0 mir-1014 96 1890 0.0507936507937 0.0
mir-1013 27 1737 0.0155440414508 6.87786055886e-09 mir-991 23 1700 0.0135294117647 5.17557054885e-06 chinmo 24 2123 0.0113047574187 9.09753678291e-06
mir-1000 129 1752 0.0736301369863 0.0 mir-1014 121 1890 0.0640211640212 0.0 mir-985 149 2484 0.0599838969404 0.0
mir-1013 27 1737 0.0155440414508 6.87786055886e-09 mir-991 23 1700 0.0135294117647 5.17557054885e-06 chinmo 24 2123 0.0113047574187 9.09753678291e-06
Up
mir-1011 103 1149 0.0896431679721 0.0 mir-1000 149 1752 0.0850456621005 0.0 mir-1006 133 1651 0.0805572380376 0.0
Down
chinmo Chronologically inappropriate morphogenesis 40 2123 0.0188412623646 1.7098976679e-09 sens senseless 92 6318 0.0145615701171 3.18664344912e-09 mir-1013 34 1737 0.0195739781232 9.52514974673e-08
Up
mir-1000 137 1752 0.078196347032 0.0 mir-991 128 1700 0.0752941176471 0.0 mir-1014 140 1890 0.0740740740741 0.0
Down
mip120 94 4885 0.0192425793245 3.84525744579e-13 E2f2 53 2279 0.0232558139535 1.03117514527e-12 Chro 122 6947 0.0175615373543 1.30478960969e-12
Up
mir-1000 116 1752 0.0662100456621 0.0 mir-1006 109 1651 0.0660205935796 0.0 mir-1002 86 1228 0.0700325732899 0.0
Down
E2f2 39 2279 0.0171127687582 5.55111512313e-16 sens 78 6318 0.0123456790123 7.77156117238e-16 Chro 82 6947 0.0118036562545 1.33226762955e-15
Up
mir-1000 92 1752 0.0525114155251 0.0 mir-283 71 1377 0.0515613652869 0.0 mir-315 113 2513 0.0449661758854 0.0
Down
sens 74 6318 0.0117125672681 1.04789177335e-10 mir-1013 27 1737 0.0155440414508 9.56715134337e-08 chinmo 33 2123 0.0155440414508 1.00435025907e-07
Up
mir-iab-4 124 1319 0.0940106141016 0.0 mir-1000 154 1752 0.087899543379 0.0 mir-1006 142 1651 0.0860084797093 0.0
Down
mip130 130 97 2783 0.0348544735897 0.0 sens 165 6318 0.0261158594492 0.0 Cp190 186 7080 0.0262711864407 0.0
Up
mir-iab-4 118 1319 0.0894617134193 0.0 mir-33 132 1648 0.0800970873786 0.0 mir-4 112 1267 0.0883977900552 0.0
Down
sens 78 6318 0.0123456790123 8.88678020061e-13 Cp190 81 7080 0.0114406779661 6.65464350291e-11 E2f2 2 34 2279 0.0149188240456 1.26859078797e-10
Up
mir-375 67 1267 0.0528808208366 0.0 su(Hw) 113 1487 0.0759919300605 0.0 mir-315 113 1487 0.0759919300605 0.0
Down
chinmo 39 2123 0.0183702308055 4.35596558823e-11 sens 80 6318 0.0126622348845 1.38819067352e-10 mir-1013 32 1737 0.0184225676454 1.00726477159e-09
Gene expression profiles were loaded into Denigma and analysed with the Expressions Application with expression ratio of 2-fold and p-value cutoff 0.05.
DR leads commonly to the upregulated lifespan factors of the following lifespan factors: Sh, to, Prm, Rh2, Rbp9, and CG6048.
Commonly enriched functional clusters among the upregulated transcripts are: ion transport, vision, alternative splicing, disulfide bond, contractile fiber, calcium.
Timeless mutants exhibit a suppressed lifespan extension by DR. A significant reduction in the lifespan is only prevalent under DR. Comparing the time on DR to wild-type on Dr should provide insights why time mutation shortens the lifespan.
Theoretically should be possible to infer from the gene expression data some valuable insights on whats going on upon DR in wild type and timeless mutant.
There is evidence that the circadian clock is quite important for the lifespan-extending effect of DR. This was previously detected mammals and here we observed similar trends in fruit fly.
One hypothesis that is that DR alters the oscillation so the that endogenous processes that counteract aging like repair are more efficient and processes that lead to age-related changes are suppressed.
There are clear trends in the interaction networks of the circadian DR signatures. Processes like DNA repair and autophagy are downregulated upon DR in timeless mutant (if compared with wild-type), while redox process associated with oxidative stress are upregulated. Genes associated with circadian rhyhtm and rhytmic processes are themselves downregulated in timeless mutants under DR.
