BACKGROUND
the glucose-6-phosphatase  <dig>  enzyme catalyses the hydrolysis of glucose-6-phosphate, produced from glycogen or gluconeogenic precursors, into inorganic phosphate and glucose, which are then released in the extracellular compartment.
g6pc plays a crucial role in glucose homeostasis by completing the final step of gluconeogenesis and glycogenolysis.
the deregulation of g6pc expression thus contributes to several physiological pathologies such as glycogen storage disease type 1a   <cit>  or type  <dig> diabete   <cit>  in mammals.
in these animals, this enzyme is encoded by the g6pc gene  which is mainly expressed in liver, intestinal mucosa, kidney and pancreatic ß-cell  <cit> .
it was recently demonstrated in teleost fish that g6pc is encoded by  <dig> to  <dig> duplicated genes depending on the genus/phylogenetic lineage  <cit> .
since the insightful suggestion of ohno in the 70’s  <cit> , whole genome duplication  has been considered to play a major role in the evolution of vertebrates.
indeed, the origin and evolution of new genes following wgd can provide new molecular and cellular functions, and play an important role in phenotypic variability and genome evolution  <cit> .
after wgd, the duplicated genes can encounter different fates: they can be lost or fixed and maintained with three distinct outcomes, i.e., neofunctionalisation, subfunctionalisation, and conservation of function  <cit> .
thus, gene duplications have played important roles in the evolution of several biological processes and/or functions including reproduction, development of the human brain, behavioural control as well as the metabolic responses of organisms  <cit> .
such an adaptive novelty was suggested in rainbow trout in which  <dig> genes encoding the glucose-6-phosphatase were identified  <cit> .
indeed, two  of these five genes are atypically up-regulated by dietary carbohydrates  <cit>  in the liver of this fish but also by glucose or insulin in vitro  <cit> .
together with an analysis of the global g6pc enzyme activity, these results strongly suggest a role of the g6pc duplicated genes in the establishment of the glucose-intolerant phenotype in trout and thus in the modulation of the metabolic response to a high dietary carbohydrate supply  <cit> .
however, but legitimately on account of its central role in the intermediary metabolism, these conclusions focused only on the liver and are restricted to a single species.
to better understand how the presence of additional copies of these genes contributes to phenotypic evolution in different organisms, a closer examination of their general distributions and characteristics in different genomes will be informative  <cit> .
in addition, to assess more comprehensively the functional importance of duplicated genes in driving phenotypic diversity in vertebrates, it will be interesting to take an evolutionary perspective, i.e.
related to their ancestral genes.
in order to conduct such an analysis, the evolutionary history of g6pc has to be thoroughly described.
indeed, during vertebrates’ evolution several rounds of whole genome duplication  occurred  <cit> : one at the emergence of chondrichthyes , one at the radiation of teleosts  and one at the radiation of salmonids .
these wgd duplications led to gene duplication.
moreover, duplicated genes can also originate from small-scale duplications  which can produce different kind of adaptations compared to wgd  <cit> .
the increasing number of sequenced genomes, extending the taxons sampling, can help providing new insights into the evolutionary history and the origin of genes  and the encoded protein function  <cit> .
in this context, the present study first aimed at revisiting and clarifying the evolutionary history of g6pc encoding genes among vertebrates, taking into consideration all genomes sequenced and indexed in ensembl genome browser .
since comparative studies of gene expression are critical for establishing links between divergent gene expression and divergence of a particular phenotype, we thus proposed to examine the evolution of the spatial g6pc expression pattern in vertebrates using the powerful phylofish database  <cit> .
indeed, this database provides a transcriptomic repertoire of  <dig> tissues and organs in  <dig> different ray-finned fish species including two holosteans  and  <dig> teleosts among which  <dig> salmonids.
by supplementing these data by in vivo analysis of g6pc in a jawless fish  and by the in silico expression pattern of several sarcopterygii g6pc  provided by the gene-annotation portal biogps  <cit>  and by the expression atlas  <cit> , we intend to provide new insights into the expression domains of g6pc in vertebrates.
the latter results should provide new trails of investigations  to better understand the atypical regulation of duplicated g6pc genes by dietary carbohydrates in salmonid  <cit> .
RESULTS
phylogenetic and synteny analysis of g6pc evolutionary history among vertebrates
by analysing vertebrate genomes available in ensembl , we found one gene related to the glucose-6-phosphatase encoding gene  in lamprey, a jawless vertebrate;  <dig> in spotted gar, a holostei; between  <dig> and  <dig> in amphibians, reptiles and owes and one in mammals, and  <dig> to  <dig> in teleosts  .
based on the automatic annotation provided in ensembl, g6pc appeared to be lost in duck, chicken, platypus, opossum, tasmania devil, armadillo, pig, tree shrew and orangutan.
using blast and syntenic  approaches we found one g6pc gene in each of these species .
moreover, we identified two genomic sequences, scaffold  <dig> and contig  <dig>  bearing two g6pc related genes in the elephant shark  genome , a cartilaginous fish .
the size of the scaffold/contig bearing these sequences was not sufficient to cover the whole gene length.
the length of the deduced amino acid sequence was therefore not sufficient for phylogenetic analysis .
however, these truncated elephant shark sequences were submitted to a blast search against the spotted gar genome in the ensembl database.
the hit with the highest score matched the spotted gar g6pca when blasting the scaffold  <dig> sequence  and spotted gar g6pcb gene when blasting the contig  <dig> sequence  .
we then selected representative ensembl species for each class of vertebrate as well as sequences previously found in the rainbow trout genome  <cit> , a salmonid fish, and performed a phylogenetic analysis.
this analysis showed that the g6pc gene identified in lamprey stemmed from the base of the phylogenetic tree .
for the spotted gar, one g6pc gene  rooted with teleosts g6pca genes while the other  was orthologous to both g6pcb <dig> and g6pcb <dig> genes previously described in teleosts  <cit> , defining them as g6pca and g6pcb, respectively.fig.
<dig> phylogenetic analysis of glucose-6-phosphatase encoding genes in vertebrates.
phylogenetic analyses were performed using mega package version  <dig> software  <cit> , as previously described  <cit> .
the phylogenetic tree was built by the neighbor-joining  method.
the reliability of the inferred tree was estimated by the bootstrap method with  <dig> replications.
mammalian and teleosts g6pc <dig> protein sequences were used to root the tree.
all accession numbers  are specified in parentheses.
sarcopterygii species are written in orange and actinopterygii species in green
this phylogenetic tree also revealed that all g6pc identified in sarcopterygii  and in the coelacanth grouped together and were orthologous to g6pcb related genes in holostei and teleosts.
we then performed a synteny analysis to clarify the evolutionary history of g6pc in vertebrates.
the chromosome bearing g6pc in lamprey was too short to provide relevant synteny information.
however, our analysis, in addition to the one previously proposed by marandel et al.
in teleosts, showed that g6pc genes found in sarcopterygii were all included in a syntenic group conserved across vertebrates .
to identify potential remnants of the g6pca gene in sarcopterygii, the spotted gar g6pca cdna sequence was submitted to a blat search  against sequenced genomes of sarcopterygii.
unfortunately, we were unable to find any significant hit in analysed genomes.
thus, in order to confirm the identity of the only g6pc gene conserved in sarcopterygii and to strengthen our phylogenetic analysis, we performed a protein percentage identity matrix and an alignment.
the identity matrix  and associated alignment  confirmed that g6pc proteins in sarcopterygii shared higher sequence identity with the spotted gar g6pcb than with its g6pca paralog.
remarkably g6pc protein, whatever the orthologous gene by which it is encoded  or the species considered, shared high sequence conservation around the binding  and the active  sites of the enzyme .
it is also noteworthy that the exon/intron structure of g6pc genes was conserved among osteichthyes evolution as all g6pc genomic structures displayed  <dig> exons and  <dig> introns  .fig.
<dig> conserved synteny around the g6pc loci in vertebrates.
data were collected with genomicus software version  <dig> , and we annotated g6pc genes according to our phylogenetic analysis following zfin nomenclature guidelines.
the teleosts synteny was modified from marandel et al.
.
remnants of g6pca and g6pcb <dig> were identified by red crosses and stars, respectively.
ch., chromosome; sc., scaffold, cont., contig; 2r, second whole genome duplication; ts3r, teleost-specific whole genome duplication; ss4r, salmonid-specific whole genome duplication
a blat analysis was also conducted in teleosts, in order to identify remnants of genes suspected to have been lost in different orders   <cit> .
we identified one hit  on the zebrafish chromosome  <dig>  and one on the rainbow trout scaffold_ <dig>  for g6pca remnant but none on the scaffold_ <dig> or the scaffold_ <dig> of trout.
in addition, one hit  on the medaka chromosome  <dig>  and one on the tetraodon chromosome un_random  were found for g6pcb <dig> remnant.
evolution of g6pc expression domains in vertebrates
the phylofish database  <cit> , a unique resource providing comprehensive expressed gene repertoires in  <dig> ray-finned fish species, was used to explore the evolution of the spatial expression of g6pc genes.
in order to have an overall view across the vertebrate subphylum, we also supplemented this analysis by rt-qpcr in lamprey and by in silico data analysis for several sarcopterygii  provided by the gene-annotation portal biogps  <cit>  and by the expression atlas  <cit> .
the expression domains of g6pc genes of representative species are presented in fig.
<dig> and data obtained from phylofish in the  <dig> ray-finned fish and in other sarcopterygii are displayed in additional file 3: figures s <dig> and additional file 4: figure s <dig>  respectively.fig.
<dig> tissue expression profiles of g6pc genes in vertebrates.
excepted for lamprey, relative expression of g6pc genes was calculated as the percentage of the maximum rpkm  value per species.
these data were obtained from biogps database for the mouse and from phylofish database for other species.
relative abundance of g6pc mrna in lamprey was established by rt-qpcr analysis and expressed as means ± se
our results showed that g6pc was preferentially expressed in the brain and in the liver of lamprey .
based on g6pca expression patterns in phylofish species, we observed that this gene was mainly expressed in the liver of holostei  and teleosts.
in addition, g6pca was also highly expressed in intestine and kidney of the spotted gar, and to a lower extend in zebrafish and medaka.
as for g6pcb genes, we found in the biogps and the expression atlas databases that g6pcb in sarcopterygii  was also preferentially expressed in liver, intestine and kidney whereas in holostei, in addition to these latter three organs, it was mainly expressed in heart.
g6pcb <dig> was also showed to be preferentially expressed in heart in cypriniformes , characiformes  and esociformes  as well as in salmonid .
in salmonid, g6pcb <dig> duplicated genes were also highly expressed in brain and in a lesser extent in all other analysed organs.
in contrast, but similarly to g6pca, g6pcb <dig> was preferentially expressed in liver, intestine and kidney in all teleost species excepted for salmonids in which g6pcb <dig> duplicated genes were mainly expressed in the liver.
expression pattern of g6pc genes in brain and heart of rainbow trout fed a high carbohydrate diet
as we found that g6pcb <dig> genes were preferentially expressed in brain and heart in salmonids, we investigated the expression pattern of g6pc genes in heart and different areas of brain of rainbow trout fed diets without  or with a high amount  of carbohydrates.
this analysis revealed that g6pcb2a and g6pcb2b mrna levels were higher in the telencephalon of trout fed the highcho diet compared to fasted trout .
the same expression pattern was also monitored in the hindbrain for g6pcb2b.
moreover, g6pcb2b mrna level was higher in the telencephalon of trout fed the highcho diet compared to trout fed the nocho diet .fig.
<dig> relative expression of g6pc genes in brain regions and heart of rainbow trout.
relative abundance of g6pc paralogs in fasted trout , trout fed with the nocho  or the highcho  diet.
data are expressed as means ± se .
different letters over bars indicate significant differences between conditions .
tel, telencephalon; hyp, hypothalamus; mid, midbrain; hind, hindbrain
discussion
g6pc duplications happened by successive rounds of wgd during vertebrate evolution.
all the results related to the evolution of g6pc in vertebrates and obtained in this study are summarised in fig.
<dig> fig.
<dig> summary of the evolutionary history and spatial expression patterns of g6pc genes in vertebrates
the first critical step in elucidating the evolutionary history of g6pc gene was to accurately identify orthologous genes in investigated species.
our phylogenetic and synteny analyses demonstrated that there was only one g6pc gene in lamprey, a jawless fish, but also only one gene in sarcopterygii whereas at least two orthologous g6pc genes were found in chondrichthyes and actinopterygii.
while it is commonly accepted that 1/2r occurred before the divergence of chondrichthyes  <cit> , these results thus strongly suggested that g6pc duplicated before or around osteichthyes/chondrichthyes radiation, giving rise to g6pca and g6pcb probably as a consequence of the 2r.
our phylogenetic analysis led to the conclusion that g6pca was lost after this duplication in sarcopterygii.
the broad view of g6pc evolution including all vertebrates presented here improved the understanding of the timing of g6pc duplication.
indeed, in a previous study  <cit>  focused on teleosts, we only considered sarcopterygii species as outgroup to elucidate g6pc evolution in this infraclass.
we thus concluded that, due to the presence of only one g6pca gene and two g6pcb genes in the genome of several teleosts, a duplication of g6pc occurred before or around teleost radiation followed by an additional duplication of g6pcb that occurred at least in the genome of the common ancestor of the euteleosts, salmonids, and ostariophys.
by the identification of g6pca remnants in zebrafish and rainbow trout and g6pcb <dig> remnants in medaka and tetraodon, our present results also showed that g6pca and g6pcb duplicated as a consequence of the ts3r.
these results also showed that one g6pca duplicate was lost in teleost genomes and confirmed the findings previously proposed by marandel et al.
that g6pcb <dig> was lost in percomorpha.
in conclusion, together with the finding previously reported in teleosts  <cit> , our results strongly suggest that g6pc diversity originates, at least in part, from successive whole genome duplications from 2r to ss4r.
in addition, duplicated g6pcb genes found in xenopus , finch and anole lizard probably arose from a lineage-specific duplication.
g6pc functionally diverged among vertebrates through sub- and neo-functionalisation of their spatial pattern of expression
the emergence of new genes through duplication is now considered as a driver process of evolution.
indeed, these new genes are genetic material available for supporting the rise of new molecular and cellular functions, and can play an important role in phenotypic variability .
duplicated genes fixed in a genome can conserve the function of the ancestral gene or functionally diverge  <cit> .
collected information about ancestral state is critical for the classification of this divergence into sub- and neofunctionalisation  <cit> .
we thus analyzed the spatial expression pattern of g6pc genes in vertebrates completing information obtained from the phylofish, the biogps and the expression atlas databases by rt-qpcr analysis in lamprey .
our results indicate that, following 2r, the g6pc expression in the brain found in lamprey was lost in tetrapods as well as in holostei whereas the hepatic expression of g6pca and g6pcb remained.
by contrast, intestine, kidney, and heart were identified as new territories of g6pca and g6pcb expression in both tetrapods and holostei.
however, a g6pc activity was previously described in kidney   <cit>  as well as in brain of mammals  <cit> .
the missing result in databases concerning g6pc expression in the brain of at least mammals  was probably due to the faint expression of this gene in this tissue  <cit> .
for other tissues, our results in lamprey  and tetrapods were in accordance with published works  <cit> .
together with previous published data, our results suggest that after the 2r, g6pc duplicates underwent neofunctionalization in heart and intestine.
after the ts3r, our results showed that g6pca remained mainly expressed in intestine, liver and kidney as well as g6pcb <dig>  in contrast, g6pcb <dig> was preferentially found expressed in the heart in species even though the expression in intestine, liver and kidney was anyway detected.
to our knowledge, g6pcb <dig> expression pattern has never been studied in non-salmonid teleosts, thus our results strongly suggested for the first time that g6pcb <dig> and g6pcb <dig> were subfunctionalised after ts3r.
moreover, data collected from the phylofish database were in favor of the loss of the expression of g6pc genes in brain of non-salmonid teleosts after the ts3rtgd.
as the functional role of g6pc in brain and heart has never been elucidated, further functional studies are required to investigate the possible functional schemes of such a loss as well as of the loss of g6pcb <dig> in percomorpha species.
finally, in salmonids g6pca was almost exclusively expressed in the liver as well as g6pcb1a whereas g6pcb1b mrna was found mainly in intestine in addition to liver.
by contrast, g6pcb2a and g6pcb2b displayed a ubiquitous expression but were mostly found in brain and heart whereas they were faintly expressed in liver.
as g6pcb <dig> was not found in the brain of non-salmonid teleosts, this finding was in favor of a neofunctionalisation of g6pcb <dig> genes in the brain of salmonids.
however, we cannot rule out the possibility that the new expression of g6pc gene in brain was gained independently in lamprey and salmonid lineages.
in conclusion, this spatial expression pattern analysis showed that g6pc diverged during vertebrate evolution mainly through sub- or neo-functionalization at lest in terms of expression domains.
our results also showed that important protein domains  promoting the activity of the enzyme were conserved during vertebrate evolution as well as the exon/intron structure of g6pc genes.
together, our findings suggest that g6pc orthologs/paralogs diversified at the level of their regulation as previously supposed by epigenetic investigations carried out in salmonids  <cit> .
in addition, the most surprising result of our study was the report of g6pc mrna expression in heart and brain.
indeed, these two organs need glucose as fuel energy to sustain their metabolic requirements, they are thus considered as glycolytic rather than gluconeogenic organs.
even if g6pc activity was previously detected in heart and brain of mammals  and brain of lamprey , its role remains poorly understood.
an unexpected result was that salmonid g6pcb <dig> paralogs were found mostly expressed in brain and heart and poorly found in liver.
indeed these genes were previously shown to display an atypical up-regulation of their mrna levels in the liver of trout fed a high carbohydrate diet  <cit> .
in order to better understand the role of these genes we thus investigated their expression patterns in different brain areas and in heart in trout fed with or without dietary carbohydrates.
g6pcb2b gene was up-regulated by dietary carbohydrates the in telencephalon of trout
the discovery of new g6pc encoding genes, i.e.
g6pcb <dig> genes, in salmonids  <cit>  and the present results, showing their neofunctionalisation and preferential expression in brain and heart, suggested new function of these genes in these tissues.
we thus investigated their expression patterns in different areas of brain and in heart of fasted trout and of trout fed a high or a no carbohydrate diet.
our results showed that there were no differences in mrna levels of g6pc paralogs in heart whatever the nutritional status of trout.
in brain regions, we observed that g6pca and duplicated g6pcb <dig> remained stable whereas g6pcb2b increased in telencephalon of rainbow trout fed the carbohydrate-enriched diet.
this result suggested the functionalisation of g6pcb2b genes in this brain area related to the content of carbohydrates in the diet.
what functions may cover this change in enzyme expression?
there is evidence for possible relationships of g6pc/endogenous glucose production pathway with glucose homeostasis.
in rainbow trout brain the presence of glucosensing mechanisms was demonstrated in hypothalamus and hindbrain  and they relate to the control of food intake as well as to counter-regulatory responses to changes in circulating levels of glucose .
these mechanisms result in enhanced glycolytic potential in response to increased levels of extracellular glucose.
since hindbrain and probably telencephalon are glucosensing areas in this species  <cit>  we would expect a priori a decrease in the mrna levels of g6pc genes in response to increased levels of carbohydrates in the diet.
however, this response did not occur in the present experiment thus not supporting that the responses observed relate to glucosensing capacity.
this is further supported by the fact that hypothalamus  did not show any response to changes in dietary carbohydrate content.
therefore, the changes observed in response to carbohydrate intake may relate to functions different than those elicited by canonical glucosensors on food intake regulation and counter-regulatory mechanisms .
several studies reported the presence of active g6pc enzyme in brain of vertebrates including agnathes  <cit> , elasmobranchs  <cit> , tetrapods , and teleosts  <cit> .
in lampreys, available studies suggest that this tissue could be, in a certain extent, autonomous from glucose supplied through the blood as demonstrated by endogenous glucose production either from gluconeogenesis or glycogenolysis, a process in which glucose-6-phosphatase action is involved  <cit>  thus suggesting a possible function of g6pc.
in teleosts, levels of glycogen in brain are considerably lower than in lamprey even though the enzymes involved in gluconeogenesis and glycogen metabolism are also present and functional as previously demonstrated in rainbow trout  <cit> .
the role of gluconeogenesis in teleost brain is however still an enigma though it could relate to glucose production under conditions of limited glucose supply such as under food deprivation.
in fact, studies in food-deprived rainbow trout  <cit>  showed increased glycogenolytic capacity and thus presumably g6pc activity in the whole brain.
changes observed in the present study in response to increased dietary carbohydrate levels are not consistent with this hypothesis since decreased rather than increased expression of g6pc is expected.
these findings suggest that g6pcb <dig> may endorse another function in the brain and be involved in specific unknown process related to specific functions of the telencephalic  area.
finally, the present g6pcb2b expression pattern in telencephalon was previously observed in the liver of trout subjected to the same experimental plan  <cit> .
the up-regulation of this gene in trout fed a high carbohydrate diet mimicked expression profile of gckb in the telencephalon , one of the two ohnologous genes that encodes for the first enzyme of the glycolysis pathway , as well as what was observed in the liver.
based on this, it was hypothesised that a futile glucose/glucose-6-phosphate cycling, involving g6pc and gck, may take place in the liver in order for example to protect cells from oxidative stress  <cit> .
the existence of such a futile cycle in the brain remains under debate in mammals  but has never been studied in teleosts to date.
in conclusion, g6pcb2b was atypically up-regulated by dietary carbohydrates in a localized brain area.
its role however remained questioning and further investigations such as knock-out of g6pcb <dig> genes are needed to better understand the function they acquired after duplication.
CONCLUSIONS
in conclusion, the present study proposes for the first time a thorough analysis of the evolutionary history of glucose-6-phosphatase genes in vertebrates.
our results showed that the diversity of g6pc genes results from successive rounds of whole genome duplication that occurred in vertebrates  followed by the loss of some duplicates in specific lineages.
we also report that the spatial expression pattern of duplicated genes significantly diverged in the different vertebrate lineages investigated.
finally, this evolution of spatial expression patterns of g6pc genes in vertebrates demonstrated that g6pcb <dig> genes, which were shown to be atypically regulated by dietary carbohydrates in salmonid liver, were mainly expressed in brain and heart.
we showed that g6pcb2b was up-regulated by dietary carbohydrates in telencephalon of rainbow trout and not in other brain areas.
the increased capacity for endogenous glucose production suggested by this change in expression is not compatible with glucosensing and may relate to specific unknown processes specific of these brain areas.
together, our results strongly suggest that the fixation and the divergence of g6pc duplicated genes during vertebrate evolution may lead to adaptive novelty and probably to the emergence of particular phenotypes related to glucose homeostasis.
as previously proposed for the glycolytic pathway  <cit> , gluconeogenesis should have thus subsequently evolved by gene duplication and divergence of g6pc.

 the glucose-6-phosphatase enzyme catalyses the hydrolysis of glucose-6-phosphate, produced from glycogen or gluconeogenic precursors, into inorganic phosphate and glucose, which are then released in the extracellular compartment. g6pc plays a crucial role in glucose homeostasis by completing the final step of gluconeogenesis and glycogenolysis. the deregulation of g6pc expression thus contributes to several physiological pathologies such as glycogen storage disease type 1a or type diabete in mammals. in these animals, this enzyme is encoded by the g6pc gene which is mainly expressed in liver, intestinal mucosa, kidney and pancreatic ß-cell. it was recently demonstrated in teleost fish that g6pc is encoded by to duplicated genes depending on the genus/phylogenetic lineage. since the insightful suggestion of ohno in the 70’s, whole genome duplication has been considered to play a major role in the evolution of vertebrates. indeed, the origin and evolution of new genes following wgd can provide new molecular and cellular functions, and play an important role in phenotypic variability and genome evolution. after wgd, the duplicated genes can encounter different fates: they can be lost or fixed and maintained with three distinct outcomes, i.e., neofunctionalisation, subfunctionalisation, and conservation of function. thus, gene duplications have played important roles in the evolution of several biological processes and/or functions including reproduction, development of the human brain, behavioural control as well as the metabolic responses of organisms. such an adaptive novelty was suggested in rainbow trout in which genes encoding the glucose-6-phosphatase were identified. indeed, two of these five genes are atypically up-regulated by dietary carbohydrates in the liver of this fish but also by glucose or insulin in vitro. together with an analysis of the global g6pc enzyme activity, these results strongly suggest a role of the g6pc duplicated genes in the establishment of the glucose-intolerant phenotype in trout and thus in the modulation of the metabolic response to a high dietary carbohydrate supply. however, but legitimately on account of its central role in the intermediary metabolism, these conclusions focused only on the liver and are restricted to a single species. to better understand how the presence of additional copies of these genes contributes to phenotypic evolution in different organisms, a closer examination of their general distributions and characteristics in different genomes will be informative. in addition, to assess more comprehensively the functional importance of duplicated genes in driving phenotypic diversity in vertebrates, it will be interesting to take an evolutionary perspective, i.e. related to their ancestral genes. in order to conduct such an analysis, the evolutionary history of g6pc has to be thoroughly described. indeed, during vertebrates’ evolution several rounds of whole genome duplication occurred: one at the emergence of chondrichthyes, one at the radiation of teleosts and one at the radiation of salmonids. these wgd duplications led to gene duplication. moreover, duplicated genes can also originate from small-scale duplications which can produce different kind of adaptations compared to wgd. the increasing number of sequenced genomes, extending the taxons sampling, can help providing new insights into the evolutionary history and the origin of genes and the encoded protein function. in this context, the present study first aimed at revisiting and clarifying the evolutionary history of g6pc encoding genes among vertebrates, taking into consideration all genomes sequenced and indexed in ensembl genome browser. since comparative studies of gene expression are critical for establishing links between divergent gene expression and divergence of a particular phenotype, we thus proposed to examine the evolution of the spatial g6pc expression pattern in vertebrates using the powerful phylofish database. indeed, this database provides a transcriptomic repertoire of tissues and organs in different ray-finned fish species including two holosteans and teleosts among which salmonids. by supplementing these data by in vivo analysis of g6pc in a jawless fish and by the in silico expression pattern of several sarcopterygii g6pc provided by the gene-annotation portal biogps and by the expression atlas, we intend to provide new insights into the expression domains of g6pc in vertebrates. the latter results should provide new trails of investigations to better understand the atypical regulation of duplicated g6pc genes by dietary carbohydrates in salmonid.  phylogenetic and synteny analysis of g6pc evolutionary history among vertebrates by analysing vertebrate genomes available in ensembl, we found one gene related to the glucose-6-phosphatase encoding gene in lamprey, a jawless vertebrate; in spotted gar, a holostei; between and in amphibians, reptiles and owes and one in mammals, and to in teleosts. based on the automatic annotation provided in ensembl, g6pc appeared to be lost in duck, chicken, platypus, opossum, tasmania devil, armadillo, pig, tree shrew and orangutan. using blast and syntenic approaches we found one g6pc gene in each of these species. moreover, we identified two genomic sequences, scaffold and contig bearing two g6pc related genes in the elephant shark genome, a cartilaginous fish. the size of the scaffold/contig bearing these sequences was not sufficient to cover the whole gene length. the length of the deduced amino acid sequence was therefore not sufficient for phylogenetic analysis. however, these truncated elephant shark sequences were submitted to a blast search against the spotted gar genome in the ensembl database. the hit with the highest score matched the spotted gar g6pca when blasting the scaffold sequence and spotted gar g6pcb gene when blasting the contig sequence. we then selected representative ensembl species for each class of vertebrate as well as sequences previously found in the rainbow trout genome, a salmonid fish, and performed a phylogenetic analysis. this analysis showed that the g6pc gene identified in lamprey stemmed from the base of the phylogenetic tree. for the spotted gar, one g6pc gene rooted with teleosts g6pca genes while the other was orthologous to both g6pcb and g6pcb genes previously described in teleosts, defining them as g6pca and g6pcb, respectively.fig. phylogenetic analysis of glucose-6-phosphatase encoding genes in vertebrates. phylogenetic analyses were performed using mega package version software, as previously described. the phylogenetic tree was built by the neighbor-joining method. the reliability of the inferred tree was estimated by the bootstrap method with replications. mammalian and teleosts g6pc protein sequences were used to root the tree. all accession numbers are specified in parentheses. sarcopterygii species are written in orange and actinopterygii species in green this phylogenetic tree also revealed that all g6pc identified in sarcopterygii and in the coelacanth grouped together and were orthologous to g6pcb related genes in holostei and teleosts. we then performed a synteny analysis to clarify the evolutionary history of g6pc in vertebrates. the chromosome bearing g6pc in lamprey was too short to provide relevant synteny information. however, our analysis, in addition to the one previously proposed by marandel et al. in teleosts, showed that g6pc genes found in sarcopterygii were all included in a syntenic group conserved across vertebrates. to identify potential remnants of the g6pca gene in sarcopterygii, the spotted gar g6pca cdna sequence was submitted to a blat search against sequenced genomes of sarcopterygii. unfortunately, we were unable to find any significant hit in analysed genomes. thus, in order to confirm the identity of the only g6pc gene conserved in sarcopterygii and to strengthen our phylogenetic analysis, we performed a protein percentage identity matrix and an alignment. the identity matrix and associated alignment confirmed that g6pc proteins in sarcopterygii shared higher sequence identity with the spotted gar g6pcb than with its g6pca paralog. remarkably g6pc protein, whatever the orthologous gene by which it is encoded or the species considered, shared high sequence conservation around the binding and the active sites of the enzyme. it is also noteworthy that the exon/intron structure of g6pc genes was conserved among osteichthyes evolution as all g6pc genomic structures displayed exons and introns.fig. conserved synteny around the g6pc loci in vertebrates. data were collected with genomicus software version, and we annotated g6pc genes according to our phylogenetic analysis following zfin nomenclature guidelines. the teleosts synteny was modified from marandel et al.. remnants of g6pca and g6pcb were identified by red crosses and stars, respectively. ch., chromosome; sc., scaffold, cont., contig; 2r, second whole genome duplication; ts3r, teleost-specific whole genome duplication; ss4r, salmonid-specific whole genome duplication a blat analysis was also conducted in teleosts, in order to identify remnants of genes suspected to have been lost in different orders. we identified one hit on the zebrafish chromosome and one on the rainbow trout scaffold_ for g6pca remnant but none on the scaffold_ or the scaffold_ of trout. in addition, one hit on the medaka chromosome and one on the tetraodon chromosome un_random were found for g6pcb remnant. evolution of g6pc expression domains in vertebrates the phylofish database, a unique resource providing comprehensive expressed gene repertoires in ray-finned fish species, was used to explore the evolution of the spatial expression of g6pc genes. in order to have an overall view across the vertebrate subphylum, we also supplemented this analysis by rt-qpcr in lamprey and by in silico data analysis for several sarcopterygii provided by the gene-annotation portal biogps and by the expression atlas. the expression domains of g6pc genes of representative species are presented in fig.  and data obtained from phylofish in the ray-finned fish and in other sarcopterygii are displayed in additional file 3: figures s and additional file 4: figure s respectively.fig. tissue expression profiles of g6pc genes in vertebrates. excepted for lamprey, relative expression of g6pc genes was calculated as the percentage of the maximum rpkm value per species. these data were obtained from biogps database for the mouse and from phylofish database for other species. relative abundance of g6pc mrna in lamprey was established by rt-qpcr analysis and expressed as means ± se our results showed that g6pc was preferentially expressed in the brain and in the liver of lamprey. based on g6pca expression patterns in phylofish species, we observed that this gene was mainly expressed in the liver of holostei and teleosts. in addition, g6pca was also highly expressed in intestine and kidney of the spotted gar, and to a lower extend in zebrafish and medaka. as for g6pcb genes, we found in the biogps and the expression atlas databases that g6pcb in sarcopterygii was also preferentially expressed in liver, intestine and kidney whereas in holostei, in addition to these latter three organs, it was mainly expressed in heart. g6pcb was also showed to be preferentially expressed in heart in cypriniformes, characiformes and esociformes as well as in salmonid. in salmonid, g6pcb duplicated genes were also highly expressed in brain and in a lesser extent in all other analysed organs. in contrast, but similarly to g6pca, g6pcb was preferentially expressed in liver, intestine and kidney in all teleost species excepted for salmonids in which g6pcb duplicated genes were mainly expressed in the liver. expression pattern of g6pc genes in brain and heart of rainbow trout fed a high carbohydrate diet as we found that g6pcb genes were preferentially expressed in brain and heart in salmonids, we investigated the expression pattern of g6pc genes in heart and different areas of brain of rainbow trout fed diets without or with a high amount of carbohydrates. this analysis revealed that g6pcb2a and g6pcb2b mrna levels were higher in the telencephalon of trout fed the highcho diet compared to fasted trout. the same expression pattern was also monitored in the hindbrain for g6pcb2b. moreover, g6pcb2b mrna level was higher in the telencephalon of trout fed the highcho diet compared to trout fed the nocho diet.fig. relative expression of g6pc genes in brain regions and heart of rainbow trout. relative abundance of g6pc paralogs in fasted trout, trout fed with the nocho or the highcho diet. data are expressed as means ± se. different letters over bars indicate significant differences between conditions. tel, telencephalon; hyp, hypothalamus; mid, midbrain; hind, hindbrain discussion g6pc duplications happened by successive rounds of wgd during vertebrate evolution. all the results related to the evolution of g6pc in vertebrates and obtained in this study are summarised in fig.  fig. summary of the evolutionary history and spatial expression patterns of g6pc genes in vertebrates the first critical step in elucidating the evolutionary history of g6pc gene was to accurately identify orthologous genes in investigated species. our phylogenetic and synteny analyses demonstrated that there was only one g6pc gene in lamprey, a jawless fish, but also only one gene in sarcopterygii whereas at least two orthologous g6pc genes were found in chondrichthyes and actinopterygii. while it is commonly accepted that 1/2r occurred before the divergence of chondrichthyes, these results thus strongly suggested that g6pc duplicated before or around osteichthyes/chondrichthyes radiation, giving rise to g6pca and g6pcb probably as a consequence of the 2r. our phylogenetic analysis led to the conclusion that g6pca was lost after this duplication in sarcopterygii. the broad view of g6pc evolution including all vertebrates presented here improved the understanding of the timing of g6pc duplication. indeed, in a previous study focused on teleosts, we only considered sarcopterygii species as outgroup to elucidate g6pc evolution in this infraclass. we thus concluded that, due to the presence of only one g6pca gene and two g6pcb genes in the genome of several teleosts, a duplication of g6pc occurred before or around teleost radiation followed by an additional duplication of g6pcb that occurred at least in the genome of the common ancestor of the euteleosts, salmonids, and ostariophys. by the identification of g6pca remnants in zebrafish and rainbow trout and g6pcb remnants in medaka and tetraodon, our present results also showed that g6pca and g6pcb duplicated as a consequence of the ts3r. these results also showed that one g6pca duplicate was lost in teleost genomes and confirmed the findings previously proposed by marandel et al. that g6pcb was lost in percomorpha. in conclusion, together with the finding previously reported in teleosts, our results strongly suggest that g6pc diversity originates, at least in part, from successive whole genome duplications from 2r to ss4r. in addition, duplicated g6pcb genes found in xenopus, finch and anole lizard probably arose from a lineage-specific duplication. g6pc functionally diverged among vertebrates through sub- and neo-functionalisation of their spatial pattern of expression the emergence of new genes through duplication is now considered as a driver process of evolution. indeed, these new genes are genetic material available for supporting the rise of new molecular and cellular functions, and can play an important role in phenotypic variability. duplicated genes fixed in a genome can conserve the function of the ancestral gene or functionally diverge. collected information about ancestral state is critical for the classification of this divergence into sub- and neofunctionalisation. we thus analyzed the spatial expression pattern of g6pc genes in vertebrates completing information obtained from the phylofish, the biogps and the expression atlas databases by rt-qpcr analysis in lamprey. our results indicate that, following 2r, the g6pc expression in the brain found in lamprey was lost in tetrapods as well as in holostei whereas the hepatic expression of g6pca and g6pcb remained. by contrast, intestine, kidney, and heart were identified as new territories of g6pca and g6pcb expression in both tetrapods and holostei. however, a g6pc activity was previously described in kidney as well as in brain of mammals. the missing result in databases concerning g6pc expression in the brain of at least mammals was probably due to the faint expression of this gene in this tissue. for other tissues, our results in lamprey and tetrapods were in accordance with published works. together with previous published data, our results suggest that after the 2r, g6pc duplicates underwent neofunctionalization in heart and intestine. after the ts3r, our results showed that g6pca remained mainly expressed in intestine, liver and kidney as well as g6pcb in contrast, g6pcb was preferentially found expressed in the heart in species even though the expression in intestine, liver and kidney was anyway detected. to our knowledge, g6pcb expression pattern has never been studied in non-salmonid teleosts, thus our results strongly suggested for the first time that g6pcb and g6pcb were subfunctionalised after ts3r. moreover, data collected from the phylofish database were in favor of the loss of the expression of g6pc genes in brain of non-salmonid teleosts after the ts3rtgd. as the functional role of g6pc in brain and heart has never been elucidated, further functional studies are required to investigate the possible functional schemes of such a loss as well as of the loss of g6pcb in percomorpha species. finally, in salmonids g6pca was almost exclusively expressed in the liver as well as g6pcb1a whereas g6pcb1b mrna was found mainly in intestine in addition to liver. by contrast, g6pcb2a and g6pcb2b displayed a ubiquitous expression but were mostly found in brain and heart whereas they were faintly expressed in liver. as g6pcb was not found in the brain of non-salmonid teleosts, this finding was in favor of a neofunctionalisation of g6pcb genes in the brain of salmonids. however, we cannot rule out the possibility that the new expression of g6pc gene in brain was gained independently in lamprey and salmonid lineages. in conclusion, this spatial expression pattern analysis showed that g6pc diverged during vertebrate evolution mainly through sub- or neo-functionalization at lest in terms of expression domains. our results also showed that important protein domains promoting the activity of the enzyme were conserved during vertebrate evolution as well as the exon/intron structure of g6pc genes. together, our findings suggest that g6pc orthologs/paralogs diversified at the level of their regulation as previously supposed by epigenetic investigations carried out in salmonids. in addition, the most surprising result of our study was the report of g6pc mrna expression in heart and brain. indeed, these two organs need glucose as fuel energy to sustain their metabolic requirements, they are thus considered as glycolytic rather than gluconeogenic organs. even if g6pc activity was previously detected in heart and brain of mammals and brain of lamprey, its role remains poorly understood. an unexpected result was that salmonid g6pcb paralogs were found mostly expressed in brain and heart and poorly found in liver. indeed these genes were previously shown to display an atypical up-regulation of their mrna levels in the liver of trout fed a high carbohydrate diet. in order to better understand the role of these genes we thus investigated their expression patterns in different brain areas and in heart in trout fed with or without dietary carbohydrates. g6pcb2b gene was up-regulated by dietary carbohydrates the in telencephalon of trout the discovery of new g6pc encoding genes, i.e. g6pcb genes, in salmonids and the present results, showing their neofunctionalisation and preferential expression in brain and heart, suggested new function of these genes in these tissues. we thus investigated their expression patterns in different areas of brain and in heart of fasted trout and of trout fed a high or a no carbohydrate diet. our results showed that there were no differences in mrna levels of g6pc paralogs in heart whatever the nutritional status of trout. in brain regions, we observed that g6pca and duplicated g6pcb remained stable whereas g6pcb2b increased in telencephalon of rainbow trout fed the carbohydrate-enriched diet. this result suggested the functionalisation of g6pcb2b genes in this brain area related to the content of carbohydrates in the diet. what functions may cover this change in enzyme expression? there is evidence for possible relationships of g6pc/endogenous glucose production pathway with glucose homeostasis. in rainbow trout brain the presence of glucosensing mechanisms was demonstrated in hypothalamus and hindbrain and they relate to the control of food intake as well as to counter-regulatory responses to changes in circulating levels of glucose. these mechanisms result in enhanced glycolytic potential in response to increased levels of extracellular glucose. since hindbrain and probably telencephalon are glucosensing areas in this species we would expect a priori a decrease in the mrna levels of g6pc genes in response to increased levels of carbohydrates in the diet. however, this response did not occur in the present experiment thus not supporting that the responses observed relate to glucosensing capacity. this is further supported by the fact that hypothalamus did not show any response to changes in dietary carbohydrate content. therefore, the changes observed in response to carbohydrate intake may relate to functions different than those elicited by canonical glucosensors on food intake regulation and counter-regulatory mechanisms. several studies reported the presence of active g6pc enzyme in brain of vertebrates including agnathes, elasmobranchs, tetrapods, and teleosts. in lampreys, available studies suggest that this tissue could be, in a certain extent, autonomous from glucose supplied through the blood as demonstrated by endogenous glucose production either from gluconeogenesis or glycogenolysis, a process in which glucose-6-phosphatase action is involved thus suggesting a possible function of g6pc. in teleosts, levels of glycogen in brain are considerably lower than in lamprey even though the enzymes involved in gluconeogenesis and glycogen metabolism are also present and functional as previously demonstrated in rainbow trout. the role of gluconeogenesis in teleost brain is however still an enigma though it could relate to glucose production under conditions of limited glucose supply such as under food deprivation. in fact, studies in food-deprived rainbow trout showed increased glycogenolytic capacity and thus presumably g6pc activity in the whole brain. changes observed in the present study in response to increased dietary carbohydrate levels are not consistent with this hypothesis since decreased rather than increased expression of g6pc is expected. these findings suggest that g6pcb may endorse another function in the brain and be involved in specific unknown process related to specific functions of the telencephalic area. finally, the present g6pcb2b expression pattern in telencephalon was previously observed in the liver of trout subjected to the same experimental plan. the up-regulation of this gene in trout fed a high carbohydrate diet mimicked expression profile of gckb in the telencephalon, one of the two ohnologous genes that encodes for the first enzyme of the glycolysis pathway, as well as what was observed in the liver. based on this, it was hypothesised that a futile glucose/glucose-6-phosphate cycling, involving g6pc and gck, may take place in the liver in order for example to protect cells from oxidative stress. the existence of such a futile cycle in the brain remains under debate in mammals but has never been studied in teleosts to date. in conclusion, g6pcb2b was atypically up-regulated by dietary carbohydrates in a localized brain area. its role however remained questioning and further investigations such as knock-out of g6pcb genes are needed to better understand the function they acquired after duplication.  in conclusion, the present study proposes for the first time a thorough analysis of the evolutionary history of glucose-6-phosphatase genes in vertebrates. our results showed that the diversity of g6pc genes results from successive rounds of whole genome duplication that occurred in vertebrates followed by the loss of some duplicates in specific lineages. we also report that the spatial expression pattern of duplicated genes significantly diverged in the different vertebrate lineages investigated. finally, this evolution of spatial expression patterns of g6pc genes in vertebrates demonstrated that g6pcb genes, which were shown to be atypically regulated by dietary carbohydrates in salmonid liver, were mainly expressed in brain and heart. we showed that g6pcb2b was up-regulated by dietary carbohydrates in telencephalon of rainbow trout and not in other brain areas. the increased capacity for endogenous glucose production suggested by this change in expression is not compatible with glucosensing and may relate to specific unknown processes specific of these brain areas. together, our results strongly suggest that the fixation and the divergence of g6pc duplicated genes during vertebrate evolution may lead to adaptive novelty and probably to the emergence of particular phenotypes related to glucose homeostasis. as previously proposed for the glycolytic pathway, gluconeogenesis should have thus subsequently evolved by gene duplication and divergence of g6pc. 
this up-regulation is in striking contrast with expected glucosensing mechanisms suggesting that its positive response to glucose relates to specific unknown processes in this brain area. one g6pca duplicate was lost after this duplication in teleosts. g6pcb2b, one gene previously hypothesised to be involved in the glucose intolerant phenotype in trout, was unexpectedly up-regulated by carbohydrates in trout telencephalon without showing significant changes in other brain regions. the present study aims at revisiting and clarifying the evolutionary history of g6pc genes in vertebrates. the analysis of the evolution of spatial expression patterns of g6pc genes in vertebrates showed that all g6pc were mainly expressed in intestine and liver whereas teleost-specific g6pcb genes were mainly and surprisingly expressed in brain and heart. g6pca was lost after this duplication in sarcopterygii whereas both g6pca and g6pcb then duplicated as a consequence of the teleost-specific whole genome duplication. our results suggested that the fixation and the divergence of g6pc duplicated genes during vertebrates’ evolution may lead to adaptive novelty and probably to the emergence of novel phenotypes related to glucose homeostasis. glucose-6-phosphate is a key enzyme involved in the regulation of the glucose homeostasis. g6pc duplications happened by successive rounds of whole genome duplication that occurred during vertebrate evolution. g6pc duplicated before or around osteichthyes/chondrichthyes radiation, giving rise to g6pca and g6pcb as a consequence of the second vertebrate whole genome duplication.
 glucose-6-phosphate is a key enzyme involved in the regulation of the glucose homeostasis. the present study aims at revisiting and clarifying the evolutionary history of g6pc genes in vertebrates.  g6pc duplications happened by successive rounds of whole genome duplication that occurred during vertebrate evolution. g6pc duplicated before or around osteichthyes/chondrichthyes radiation, giving rise to g6pca and g6pcb as a consequence of the second vertebrate whole genome duplication. g6pca was lost after this duplication in sarcopterygii whereas both g6pca and g6pcb then duplicated as a consequence of the teleost-specific whole genome duplication. one g6pca duplicate was lost after this duplication in teleosts. similarly one g6pcb duplicate was lost at least in the ancestor of percomorpha. the analysis of the evolution of spatial expression patterns of g6pc genes in vertebrates showed that all g6pc were mainly expressed in intestine and liver whereas teleost-specific g6pcb genes were mainly and surprisingly expressed in brain and heart. g6pcb2b, one gene previously hypothesised to be involved in the glucose intolerant phenotype in trout, was unexpectedly up-regulated by carbohydrates in trout telencephalon without showing significant changes in other brain regions. this up-regulation is in striking contrast with expected glucosensing mechanisms suggesting that its positive response to glucose relates to specific unknown processes in this brain area.  our results suggested that the fixation and the divergence of g6pc duplicated genes during vertebrates’ evolution may lead to adaptive novelty and probably to the emergence of novel phenotypes related to glucose homeostasis. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords teleostsduplicated genessarcopterygiiactinopterygiitroutglucosebraininra department phasehttp://dx.doi.org/ /501100003329ministerio de economía y competitividadagl2016-74857-c3-1-rsoengas josé luis european fund for regional developmentfedersoengas josé luis issue-copyright-statement© the author 2017 