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Rejuvenates metabolism

Rejuvenates metabolism

Brett, Marina Arjona, Low-carb dietary aids Ikeda. Importantly, the metabolic Rejuvenates metabolism meatbolism not Rejuveenates and Rejuvenages rapidly switch according Rejuvenates metabolism the cellular demands, a phenomenon commonly designated as metabolic reprogramming Folmes et al. For comparison, also shown are results for young MuSCs isolated three days after injury. Muscle regeneration occurs to coincide with mitochondrial biogenesis.

Rejuvenates metabolism -

Therefore, we blocked glycolysis with 2DG and performed a detailed characterization of OB dedifferentiation at 24 hpa Figure 4B.

We began by analyzing the expression profile of OB markers that serve as a read-out of their dedifferentiated status, namely bglap and runx2. We observed that inhibition of the glycolytic influx led to a significant upregulation of bglap and to a decrease in both runx2 orthologues Figure 4C , as opposed to what happens in OB undergoing dedifferentiation in normal regenerating conditions Knopf et al.

Overall, these data indicate that blocking glycolytic influx hampers mature OB dedifferentiation, which become unable to operate as a source of pre-OB resulting in impaired pre-OB pool assembly within the blastema primordium.

A Schematic representation of pre-OBs formation during regeneration. Pre-OBs arise from OB dedifferentiation and potentially from the joint OP niche. OB dedifferentiation is correlated with inactivation of NF-ΚB and increase in Cyp26b1 activity.

B Experimental design used to inhibit glycolysis. Fish are administered, via IP injection, with vehicle PBS or 2DG, from fin amputation 0 hpa until 24 hpa. C Relative gene expression of mature and pre-OBs markers, and differentiation and dedifferentiation pathways, in the whole fin stump at 24 hpa, in 2DG treated fins compared to control condition 0 hpa.

Arrowhead indicates amputation plane. E: epidermis; b: bone; m: mesenchyme. Scale bar represents µm and 30 µm in magnified panels. See Figure 4—source data 1. To further validate our observations, we decided to evaluate whether the pathways proposed to mediate mature OB dedifferentiation where also altered upon glycolysis inhibition.

During homeostasis NF-kB pathway maintains retinoic acid RA signalling in OBs, supporting differentiation.

Upon amputation, NF-kB pathway becomes inactivated and RA is degraded through activity of the RA-degrading enzyme, Cyp26b1, thereby inducing OB dedifferentiation Mishra et al.

In addition, Bmp signaling is also considered to be a potent inducer of OB differentiation during regeneration Stewart et al. Thus, we performed qPCR analysis at 24 hpa of NF-kB target genes e. nf k biaa and nf k biab , retinoic acid degrading enzyme e. cyp26b1 , and Bmp ligands e.

bmp2a and bmp2b in control and 2DG-treated fins Figure 4C. We observed an increase in NF-kB target genes and in bmp2b , accompanied by a decrease in cyp26b1 in 2DG-treated fins.

Thus, suppression of glycolysis seems to maintain the mature OB differentiation profile and to prevent their dedifferentiation after amputation, resembling a pre-amputation scenario.

We then evaluated whether glycolysis is necessary for other aspects of OB dedifferentiation namely migration toward the stump and cell cycle re-entry. Surprisingly, by measuring the relative cell displacement of the OB population in the first segment below the amputation region after 24 hpa, we observed that 2DG administration had no effect on the ability of OB to migrate and reach the amputation zone Figure 5A—E.

This indicates that blocking glycolytic machinery has a severe impact in the number of pre-OB that re-enter the cell cycle, reducing their proliferative capacity. This inhibition of cell cycle re-entry by 2DG was also observed in the blastema primordium mesenchyme Figure 5—figure supplement 1A-F,H and the overlying epidermal cap Figure 5—figure supplement 1A-F,G.

Moreover, the decline in the capacity of pre-OB to re-enter the cell cycle and to proliferate could be correlated with defects in the activity of pathways known to be indispensable for blastema proliferation, such as, Wnt, Insulin and Fgf signaling pathways Lee et al. This is predicted based on our results showing a downregulation of wnt10a , igf2b and fgf20a in 2DG-treated caudal fins in comparison to controls Figure 5J.

We further complement and confirmed our results using 3PO, a partial glycolytic inhibitor, in bglap :EGFP transgenic fish. A—D Representative images of bglap:EGFP caudal fins at 24 hpa, treated with A—B vehicle PBS or C—D 2DG. Double white arrows indicate the anterior A and posterior P axis.

White dashed lines indicate intersegment regions. Orange dashes lines delineate the bony-ray surface. E Measurement of relative OB displacement along segment 0, below the amputation plane, at 24 hpa in fins treated with vehicle PBS or 2DG.

Scale bar represents µm and 30 µm in amplified panels. J Relative gene expression at 24 hpa in 2DG treated fins, compared to control. See Figure 5—source data 1. MatLab scripts to quantify the relative osteoblast displacement after caudal fin amputation in controls and after 2DG treatment.

To exclude any effect of glycolysis inhibition on cell survival that could interfere with our observations, we performed a TUNEL assay at 24 hpa in controls and in 2DG-treated bglap :EGFP transgenic zebrafish.

This suggests that glycolysis may support cell survival in the mesenchymal compartment, but not in the epidermis or in the pre-OB pools. Thus, blocking glycolysis is sufficient to inhibit OB dedifferentiation and cell cycle re-entry, without affecting their capacity to migrate or survive.

Taken together, our results reveal that enhancing glycolysis promotes mature OB dedifferentiation into pre-OB and enabling pre-OB and other lineages to re-acquire proliferative capacity within the blastema.

We also provide evidence that energy metabolism controls these aspects of OB response to injury through a glycolysis-dependent transcriptional regulation. Therefore, these results provide solid evidence that metabolic reprogramming toward glycolysis is a novel and powerful conductor of the cell fate changes and cell cycle re-entry preceding blastema assembly.

Based on our results so far, we demonstrated a fundamental role of glycolysis in governing OB dedifferentiation and the early stages of blastema formation. Subsequently, we aimed to investigate how prolonged inhibition of glycolysis would interfere with blastema organization and with de novo OB formation Figure 6A.

At 48 hpa, the blastema is subdivided in a patterning zone PZ and in a proximal PB and distal DB compartments.

These regions are characterized by distinct OB subtypes, based on their maturation and proliferative state, exhibiting a proximal-distal hierarchical and overlapping distribution Wehner and Weidinger, ; Iovine, ; Nechiporuk and Keating, ; Poss et al. To determine how glycolysis affects the general distribution of OB subtypes within the blastema, we exposed runx2 :EGFP and osx :mCherry zebrafish to 2DG treatment through the first 48 hpa Figure 6A.

We observed that prolonged 2DG-treatment caused a severe abrogation of blastema organization Figure 6—figure supplement 1A-H , with a strong reduction in osx Figure 6—figure supplement 1B,F,D,H and runx2 Figure 6—figure supplement 1C,D,G,H when compared to control fins, indicating that 2DG administration strongly alters OB specific gene expression within the blastema.

Similar results were obtained when using the glycolytic inhibitor 3PO Figure 5—figure supplement 1I-O. A Experimental design used to inhibit glycolysis. Fish are administered, via IP injection, with control PBS or 2DG every 12 hr, from fin amputation 0 hpa until 48 hpa.

B Schematic representation of the distribution of OBs subtypes along the blastema. Arrowheads indicate amputation plane. Scale bar represents µm and 20 µm in magnified panels.

E: epidermis; B: bone; M: mesenchyme. For all graphs, statistical analysis corresponds to Mann-Whitney and Mean ± SD are displayed. See Figure 6—source data 1. Afterwards, we decided to ascertain if the proliferative abilities of each OB subtype in control and 2DG-treated fish, through a EdU 3 h-pulse assay.

As previously reported Stewart et al. Strikingly, glycolysis inhibition had a more profound impact on proliferation at 48 hpa Figure 6I—K than at 24 hpa Figure 5F—I , with both OB subtypes exhibiting a significant reduction of their proliferative capacity Figure 6H—K. We also noticed that, as observed at 24 hpa Figure 5—figure supplement 1G,H , the epidermis and mesenchyme were also affected, displaying a decrease in proliferation in 2-DG-treated fish Figure 6—figure supplement 2A-H.

Given the results obtained, the most likely interpretation is that both pre-OB and immature OB populations accumulate at the stump region since they are unable to proceed in the cell cycle and divide. Interestingly, this may indicate that, although OB dedifferentiation is compromised after blocking the glycolytic influx, pre-OB may be generated by alternative sources.

By extending 2DG treatment into the outgrowth and patterning phase of regeneration Figure 6—figure supplement 4A , we observe that while control fish are able to efficiently reconstruct the lost skeletal tissue at 7 days post-amputation dpa Figure 6—figure supplement 4C,D , in 2DG-treated animals bone regeneration was completely abolished Figure 6—figure supplement 4E, F.

Although we observe a clear effect of 2DG in inhibiting new bone regeneration, sustained 2DG exposure for 7 consecutive days Figure 6—figure supplement 4B seemed to have no impact on uninjured caudal fin morphology Figure 6—figure supplement 4G-J.

Uninjured fish subjected to 2DG possess similar caudal fin morphological parameters when compared to controls, namely the caudal fin area to width ratio Figure 6—figure supplement 4K,L and the bony-ray length to width ratio Figure 6—figure supplement 4M,N , suggesting that during this protocol 2DG does not compromise caudal fin integrity.

Overall, these data demonstrate an indispensable role of glycolysis in regulating blastema proliferation and compartmentalization with important implications for new OB generation and bone formation. OB dedifferentiation has been suggested to occur at the end of wound healing phase 0—18 hpa and during the blastema induction phase 12—24 hpa Knopf et al.

Here, by providing a deeper characterization of OB dedifferentiation, we demonstrate that this process is triggered as early as 6 hpa, in parallel with the initial wound healing response Chen et al. Moreover, our transcriptomic analysis of isolated OBs revealed a dynamic transcriptional response at 6 hpa in comparison to OBs from uninjured conditions.

This provides the first molecular characterization of OBs preceding the dedifferentiation stage, highlighting that mature OBs start changing their transcriptome earlier than expected and that the first hours after amputation are crucial for the transcriptional and phenotypic alterations leading to dedifferentiation.

The set of differentially expressed genes unveils potential new players worth revisiting in the future. Our study uncouples OB response from surrounding tissues, and addresses the early stages of fin regeneration, which are the least investigated.

In fact, most published data focus on time points from 24 hpa onwards, when wound closure has finished, blastema formation is in progress and consequently initial cell identity transitions have been dictated, potentially missing initial regulators of dedifferentiation.

Importantly, we show that at 6 hpa OB prioritise lactate-producing glycolysis, when compared to OB from uninjured fins. Additionally, we also observed a similar response at 6 hpa in the whole fin stump, corroborated by gene expression and metabolomic data.

These alterations persist at least until 24 hpa, when the blastema primordium is being assembled. Moreover, we demonstrate that this change in metabolism to enhance glycolysis occurs concomitantly with an increase in mitochondria fission.

We also show that glycolysis is indispensable to support blastema formation and regeneration. Blocking glycolysis leads to a complete blastema suppression, with a single injection of 2DG at 0 hpa being sufficient to induce aberrant blastema formation.

These results indicate that OBs and other cell lineages respond to amputation by undergoing a change in the metabolic profile that favours glycolysis Figure 7. Furthermore, glycolysis is necessary from the early onset of regeneration and the time interval when these changes in metabolism happen appears to be fundamental for the initiation of regeneration.

It is possible that early wound response signals are important to trigger changes in metabolic signature. One potentially relevant event described at this stage is reactive oxygen species ROS production Gauron et al.

ROS and cellular metabolism are tightly connected as ROS are a by-product of mitochondrial oxidation Zorov et al. ROS are shown to activate important molecules, such as HIF-1α, which has been shown to promote metabolic reprogramming toward glycolysis in other contexts Zhao et al.

It would be interesting to evaluate whether ROS production is necessary to stimulate glycolysis during caudal fin regeneration.

A In homeostasis, mature OBs reside in close contact with the bony-ray surface, secreting the collagenous bone matrix. B Upon caudal fin amputation, OBs and other cell types in the regenerating fin respond by undergoing a metabolic adaptation that stimulates glycolysis and is essential for regeneration to proceed.

Enhancing glycolytic influx promotes OB dedifferentiation, by releasing Cyp26b1 from NF-ΚB repression, and cell cycle re-entry, by interfering with the master regulation of caudal fin proliferation Fgf20a, thereby enabling OBs to act as progenitor cells.

Moreover, glycolysis is necessary to maintain the correct proliferative ability and distribution of OBs populations within the blastema, during its formation. C Glycolysis inhibition has a severe impact on OB dedifferentiation and pre-OBs pool assembly, which supports new OB formation and proliferation, ultimately leading to impaired bony-ray regeneration and suppression of blastema formation.

Our results provide the first evidence that OB and other cell types respond to amputation by engaging metabolic routes that boost lactate-producing glycolysis instead of OXPHOS, thereby acquiring metabolic traits of stem cells. It is well described that embryonic and adult stem cells exhibit metabolic preferences distinct from their differentiated progeny.

Both primed embryonic stem cells Folmes et al. This reflects an essential role of glycolysis in periods of rapid cellular growth, while oxidative metabolism is preferred in mature cells to maintain homeostasis Lunt el al.

Prioritizing glycolysis entails several advantages for rapid proliferating cells: fuels biosynthetic pathways necessary to sustain rapid cell growth and division by generating intermediaries for macromolecules synthesis e.

nucleic acids, lipids; and non-essential amino acids ; the rate ATP generation is faster through glycolysis than mitochondrial glucose oxidation DeBerardinis et al. acetylation, methylation, phosphorylation, or glycosylation Tarazona and Pourquié, ; Sun et al. The latter, extends the connection between metabolism and modulation of intracellular signaling pathways, and the epigenome, to control gene expression programs that change cell function and fate Tarazona and Pourquié, ; Sun et al.

One of the best reported examples occurs during induced-pluripotent stem cell iPSC reprogramming, in which the switch toward a glycolytic metabolism happens before the expression of endogenous reprogramming factors Cliff and Dalton, ; Folmes et al.

aerobic glycolysis in which cancer cells use primarily glycolysis, resulting in lactate production, instead of pyruvate oxidation through OXPHOS DeBerardinis et al. The metabolic changes that occur during fin regeneration share several parallels between cancer metabolism pathophysiology, namely preference for glycolysis to support proliferation and elevated levels of glutamine, an essential nutrient that supplies cancer metabolism.

Besides functioning as a precursor for nucleotides and amino acid synthesis, glutamine can be converted into glutamate, a metabolic intermediate with various fates in proliferating cells e.

protein synthesis, and incorporation into the TCA Lu et al. Interestingly, like cancer cells, our data points to an important role of glutamine and glutamate for the assembly of the blastema primordium, as our metabolome studies show an increase by and fold in glutamine and glutamate at the beginning of blastema formation, respectively.

Cancer cells also produce high levels of lactate where it is often regarded as an important oncometabolite Loeffler et al. Our studies show not only an increase in lactate during the initial stages of regeneration, but also reveal that inhibition of pyruvate conversion to lactate leads to defects in blastema formation, although milder when compared to glycolysis inhibition.

This indicates that lactate production may also contribute to proper blastema formation. The mechanisms by which the glutamine and glutamate cycle and lactate influence blastema formation should be addressed in future studies.

Unexpectedly, albeit aerobic glycolysis is known to support cancer cell migration, our results show that glycolysis is not required for mature OB recruitment and motility. It would be interesting to evaluate how alterations in glucose metabolism are regulated throughout the regenerative process, without falling into tumorigenesis.

Our data shows that enhancing glycolysis serves to adapt to the cellular demands of regeneration, but it also seems to have the power to dictate several aspects of the regeneration program, including modulation of mature OB dedifferentiation.

Previous studies have shown that OB dedifferentiation is a result of the activity of the NF-kB-RA axis Mishra et al. In homeostasis, NF-kB supports RA signalling, by blocking the expression of cyp26b1 , the RA-degrading enzyme, maintaining OB differentiation.

After amputation, NF-kB becomes inactivated and Cyp26b1 suppression is lifted, thereby protecting OB from RA and promoting their dedifferentiation Mishra et al. We show that blocking glycolysis leads to NF-kB signalling stimulation and decrease in cyp26b1 expression, providing evidence that increase in glycolytic activity precedes and is necessary to induce mature OB reprogramming into pre-OB.

In addition, our data shows that glycolysis is necessary to support pre-OB cell cycle re-entry and to sustain blastema proliferation Figure 7 and can be linked to fibroblast growth factor 20 a fgf20a , which is fundamental for blastema initiation and proliferation during regeneration Poss et al.

Mutants for fgf20a fail to form a functional blastema and are unable to proliferate Whitehead et al. Accordingly, blocking Fgf receptor 1 activity leads to a similar phenotype Lee et al.

Our work suggests that glycolysis promotes not only the expression of fgf20a , but also of other ligands that cooperate to induce fgf20a expression, such as igf2b and wnt10a Stoick-Cooper et al. Since these pathways are part of a general mechanism triggered upon amputation to stimulate proliferation, it is not surprising that glycolysis inhibition caused an overall reduction of proliferation.

In addition, the presented data indicates that glycolysis is necessary until the end of blastema formation, to generate new OBs and to maintain a proper balance between OB subtypes within the blastema Figure 7.

Decline in the total number of immature OB and in the proliferative rates of distal pre-OB and of proximal immature OB populations observed upon glycolysis inhibition, can be accounted, at least in part, by the pronounced effects of glycolysis inhibition on blastema proliferation.

Importantly, these results corroborate the idea that regeneration benefits from glycolysis both in terms of biomass generation, to support cell proliferation, and by inducing the expression of powerful mitogens, like fgf20a. Noteworthy, besides mature OBs, pre-OB can also derive from a population of OB progenitor that resides in the joint regions of the fin Ando et al.

Thus, mature OB and joint-associated progenitors may act as complementary sources that supply the pre-OB pool. In fact, we observed that glycolysis inhibition leads to a diminished number of pre-OBs before blastema formation, yet this number is back to normal after blastema formation.

Since blocking glycolysis prevented OB dedifferentiation, we could speculate that over time OB progenitors from the joints were able to replenish the pre-OB pool. Further work is needed to test this hypothesis and the impact of glucose metabolism in supporting OP activation and contribution for new OB formation.

In general terms, this study provides the first line of evidence that metabolic adaptation towards glycolysis governs mature OB dedifferentiation and blastema proliferation.

To some extent, this is mediated through glycolysis-driven changes in gene expression that allows to uncouple dedifferentiation from acquisition of proliferative capacity. Regeneration is in its essence an anabolic process. After an insult, reconfiguration of the extracellular milieu can induce metabolic adaptations that are fundamental to accommodate new cellular functions that support growth and cell fate decisions necessary for regeneration.

In line with our data, other animals with enhanced regenerative abilities, such as planarians Osuma et al. This indicates that metabolic rewiring towards glycolysis might be a conserved mechanism necessary for the regenerative process.

Importantly, changes in glucose metabolism were also shown to be necessary for regeneration of other zebrafish tissues. Like OBs in the fin, after cardiac injury, regeneration is achieved via dedifferentiation and proliferation of cardiomyocytes near the injury Kikuchi et al.

Recent studies have demonstrated that these cells switch to a glycolytic metabolism necessary for their dedifferentiation and proliferation Honkoop et al. Moreover, regeneration of the embryonic tail was shown to rely on glycolysis to support blastema formation Sinclair et al.

Glycolysis was required to fuel the hexosamine pathway Sinclair et al. Given these results and the similarities between larval tail and adult caudal fin regeneration, it would be important to examine the function of hexosamine pathway during fin regeneration.

In contrast to zebrafish, mammals possess poor capacity to perform epimorphic regeneration of complex structures, with only a few examples, such as amputated ear and digit tips Seifert and Muneoka, ; Johnson et al.

In mice models of ear and digit injuries, regeneration is impaired by OXPHOS inhibition, suggesting that in this context OXPHOS is required to mediate regeneration Shyh-Chang et al. In contrast, the MRL mice strain, which has an enhanced regenerative capacity in comparison to other mice, showed an increase of aerobic glycolysis over OXPHOS after injury of several organs Naviaux et al.

This indicates that further studies are necessary to clarify the potential role of glucose metabolism during mammalian regeneration. Regarding bone, disruption of the metabolic profile of OBs and OB sources e. mesenchymal stem cells might also have important implications for bone repair after injury and in certain pathological conditions e.

osteoporosis , as they influence OB identity status and function Lee et al. Cell metabolism can potentially be a target in the contexts of fracture healing or bone diseases, to stimulate the repair process, or to prevent OB dysfunction.

The data described here provides clear evidence that a metabolic reprogramming favouring anaerobic glycolysis occur at early stages of adult regeneration and are an integral component of the regenerative program. This is in accordance with recent regeneration studies performed in other systems and resembles many traits of the Warburg effect observed in cancer cells.

Our data indicates that OB and possibly other cell lineages favour glycolysis, to engage a specialized genetic program that enables them to act as progenitor cells. We unveil a novel and fundamental role of glycolysis in mediating mature OB dedifferentiation and cell cycle re-entry and supporting blastema assembly and proliferation.

Moreover, we have uncoupled the effects of glycolysis in mediating OB dedifferentiation from proliferation by identifying distinct downstream transcriptional targets of the glycolytic metabolism. This provides evidence that the role of glucose metabolism in regeneration is not limited to sustain macromolecule synthesis and energy production.

Overall, our findings support a notion that glucose metabolism has a powerful instructive role in regulating lineage-specific programs and generic responses to injury that induce changes in cell identity and function, crucial to prompt bone regeneration. Wild-type WT AB and transgenic zebrafish lines, namely Tg osterix:mCherryNTRo pd4 Singh et al.

Bglap:EGFP hu referred as bglap :EGFP and Tg Has. Fos:EGFP zf Knopf et al. Eef1a1:mlsEGFP referred as MLS-GFP , kindly provided by Seok-Yong Choi Kim et al.

All regeneration experiments were performed in 4—18 months-old fish and transgenics used as heterozygotes. Regenerated fins were collected from anaesthetized fish, and either processed for cryosectioning, stored in Trizol for RNA isolation, handled for Mass-spectrometry MS or for flow cytometry.

For pharmacological treatments via intraperitoneal injections IP , fish were randomized and subjected to IP injections at the designated time-points, with either 2DG Sigma-Aldrich, 0. Sigma-Aldrich, 0. IP injections were performed with an insulin syringe U G 0.

For 3PO Sigma-Aldrich and MB-6 Calbiochem treatments, compounds were diluted in DMSO and added to water from the circulating system to a final concentration of 15 µM and 2. For all experiments water was replaced daily and fish left to regenerate until the desired time-point.

For S-phase labeling, fish were subjected to caudal fin amputation and administrated with Ethynyl-2´-deoxyuridine EdU, Thermo Scientific: C, 20 µL of 10 mM solution diluted in 1 x PBS via IP injection 3 hr prior to caudal fin collection. For gene expression analysis, caudal fin composed of the regenerated tissue and one bony-ray segment proximal to the amputation plane were collected.

Pools from 4 to 5 caudal fins were used per biological replicate. Briefly, samples were homogenized in Trizol reagent Invitrogen, for cell disruption and RNA extracted as previously described Brandão et al. cDNA was synthesized from 1 μg total RNA for each sample using the Transcriptor High Fidelity cDNA Synthesis Kit Roche, , with a mixture of oligo dT and random primers.

All qPCR primers are listed in Supplementary file 2a. qPCR was performed using a FastStart Essential DNA Green Master Mix Roche, and a Roche LightCycler Cycle conditions were: 15 min pre-incubation at 95 °C and three-step amplification cycles 45 x , each cycle for 30 s at 95 °C, 15 s at 60 °C, and for 30 s at 72 °C.

In vivo Alizarin red S ARS, Sigma-Aldrich staining in the bglap :EGFP transgenic was performed prior to caudal fin amputation as previously described Bensimon-Brito et al. Briefly, fish were incubated in a 0. Caudal fins were amputated and imaged at specific time-points post-amputation.

For calcein staining in WT AB strain, caudal fins were collected at predefined time-points post-amputation and post-treatment. Fins were washed in 1 x PBS and immersed into a 0.

Afterwards, fins were washed five times in 1 x PBS, 10 min each, and left for 10 min in 1 x PBS to allow the unbound calcein to diffuse out of the tissues Brandão et al. Tissue processing for cryosections was performed as previously described Brandão et al.

Fins were then embedded in 7. Longitudinal caudal fins sections were obtained at 12 μm using a Microm cryostat Cryostat Leica CM S and slides stored at —20 °C.

For immunofluorescence on cryosections, slides were thawed for 15 min at room temperature RT , washed twice in 1 x PBS at 37 °C for 10 min and subjected to an antigen retrieval step, which consisted of a 15 min incubation at 95 °C with sodium citrate buffer 10 mM Tri-sodium citrate with 0.

Slides were then incubated in 0. For TUNEL labelling assay, cryosections were permeabilized in a sodium citrate solution 0. Slides were then incubated with primary antibodies diluted in blocking solution, ON at 4 °C for antibody details see Supplementary file 2b.

On the following day, slides were washed with PBST 6 times, 10 min each, and incubated with secondary antibodies Supplementary file 2c diluted in blocking solution, for 2 hr at RT and protected from light.

Slides were then washed three times with PBST, 10 min each, mounted with fluorescent Mounting Medium DAKO and stored at 4 °C protected from light until image acquisition. For fluorescence-activated cell sorting FACS of OB, caudal fins from bglap :EGFP transgenic line were amputated, tissue collected at specific time-points during regeneration and dissociated into single-cell suspensions.

Cell suspensions were passed through a 30 μm filter CellTricks, Sysmex and centrifuged at g for 5 min at 4 °C. Cell debris and aggregates were removed from the analysis. The fluorescence scatter Comp-FL Log::GFP was used to separate cells according to their GFP fluorescence intensity with a maximum of stringency to avoid cross-contamination.

Zebrafish WT AB strain was used as a negative control to set the GFP-positive population. The instrument was run at a constant pressure of kPa 30 psi with a µm nozzle and frequency of drop formation of approximately 40 kHz. Two and three independent biological replicates were performed for each condition at 0 and 6 hpa respectively.

For each, GFP-positive cells were collected directly into lysis and RNA stabilization buffer provided by OakLabs GmbH and vigorously shaken for 1 min. To verify the quality of the samples, cell death and purity were measured.

Samples were maintained at —80 °C until sent to OakLabs GmbH Henningsdorf, Germany for cDNA generation, microarray chip set up and data analysis. To compare the transcriptome profiles of mature OB in homeostasis to OB during dedifferentiation, a genome-wide gene expression profiling was set up using the 8x60 K ArrayXS Zebrafish platform by Agilent and performed by OakLabs GmbH Henningsdorf, Germany.

The 8x60 K ArrayXS Zebrafish represents approximately a total of around 60, zebrafish transcripts, which includes 48, coding genes, non-coding genes and 19, predicted genes annotated in the Zv9 release RNA quality was processed by Oaklabs using the Bioanalyzer Agilent Technologies , the RNA Pico Kit and a photometrical measurement with the Nanodrop spectrophotometer Thermo Fisher Scientific.

Subsequently, 2 µL of the lysis and RNA stabilization buffer, from three biological replicates of each condition 0 and 6 hpa OBs , was used for cDNA synthesis and pre-amplification using the Ovation One Direct system NuGEN. The generated cDNA was labeled with Cy3­­dCTP using the SureTag DNA Labelling Kit Agilent prior to microarray hybridisation.

Ultimately, fluorescence signals were detected by the SureScan Microarray Scanner Agilent Technologies , at a resolution of 3 µm for SurePrint G3 Gene Expression Microarrays and 5 µm for HD Microarray formats. Raw data was then subjected to processing and analysis.

Briefly, background signals were subtracted and then normalized using the ranked mean quantiles Bolstad et al. For data quality control and to identify potential outlier samples, hierarchical clustering and a principal component analysis were performed. The retrieved data was used to compare the expression profiles of OB from 6 hpa with 0 hpa.

Significantly differentially expressed genes were identified whenever p-value was lower than the 0. Transcriptome datasets analyzed on this study were submitted to NCBI Gene Expression Omnibus archive with an accession number GSE For metabolite analysis, caudal fins were collected and snap-freeze in liquid nitrogen for 5 min and diluted in a mixture containing MeOH:dH2O and an internal standard α-Aminobutyric acid AABA, 2 mM final concentration.

Samples were homogenized using tissue grinder for 5 s and using the ultrasound bath for 30 min at 4°. This was followed by sample centrifugation for 10 min at top speed at 4°, supernatant collected and stored at —20° short storage or —80 °C long storage. Samples and internal standards were analyzed in a Dionex UltiMate UHPLC Ultra-High Performance Liquid Chromatography system coupled to a heated electrospray QExactive Focus mass spectrometer Thermo Fisher Scientific, MA, USA.

Three separate LC-MS assays were applied. Glucose Sigma Aldrich and lactate Alfa Aesar detection were performed with acquisition in negative ionization mode. For every assay, four biological replicates 10 fins used per replicate were used per condition and sample injection was performed in triplicate and a volume of 5 µL was applied.

For regenerated area measurements, images of live anesthetised WT and transgenic adult caudal fins were acquired in a Zeiss Lumar V fluorescence stereoscope equipped with a Zeiss axiocam MRc camera using a 0. Images were assembled using the Fiji software Schindelin et al.

For 2-NBDG labeled WT caudal fins, images were acquired using in a Zeiss Axio Observer z1 inverted microscope for transmitted light and epifluorescence, equipped with an axiocam monochromatic camera, using an EC Plan-Neofluar 5x0. An image mosaic was acquired using transmitted light and the GFP 38HE filter.

Serial sections were acquired every 5 µms. For image processing, composite maximum intensity images and concatenation of several images along the caudal fin proximal-distal axis was performed using the Zen 3 blue software and images assembled using Fiji software Schindelin et al.

For live-imaging analysis of OB migratory dynamics in vivo, bglap :EGFP transgenic fish were anesthetised and maintained in glass bottom Petri dishes. Imaging was performed in a confocal microscope Zeiss LSM using the software ZEN B SP1.

Fish were imaged with a Plan-Neofluar 10x0. For OB motility assay, time-lapse images were acquired always in the same region of the fin, capturing the first 2 segments below the amputation plane segment 0 and segment —1 and the blastema region, and images acquired every 5 hr following amputation, during the first 25 hpa.

For assessment of OB migration in vehicle and 2DG treated fish, time-lapse images were acquired at 0 and 24 hpa. For image processing, composite maximum intensity z-stack projections were made using the Fiji software Schindelin et al. Time-lapses were assembled and computationally registered with the Fiji StackReg and MultiStackReg plugins Schindelin et al.

Immunolabeled cryosections were analyzed in confocal microscopes Zeiss LSM and Zeiss LSM controlled by ZEN B SP1 or ZEN 3. Cryosection images were acquired using a C-Apochromat 40x1. Sequential images were acquired to capture the first segment below the amputation plane and the entire regenerated region.

For image analysis and processing, composite maximum intensity z-stack projections were made using the Fiji software Schindelin et al. When required, concatenation of several images along the proximal-distal axis of the same longitudinal section was performed using the Fiji plugin 3D Pairwise Stitching Schindelin et al.

To count and measure the number of mitochondria per cell in longitudinal cryosections of individual regenerating bony-rays, we used the surface tool from IMARIS software using a mitochondria surface detail of 0. All Images were then processed using the Adobe Photoshop CS5 and Adobe Illustrator CC.

For qPCR analysis, all samples were analyzed in four to eight biological pools. For each biological pool, qPCR was performed for each target gene in three technical replicates. Measurements of total regenerated area were obtained by delineating the fin regenerated area using the Area tool in Fiji.

The regenerated area was then normalized to the corresponding total caudal fin width to avoid discrepancies related to the animal size, resulting in one measurement per animal. All quantifications were done using the Cell-counter plugin on Fiji in individual cryosections representing at least three different blastemas per animal and three to five animals per condition.

For quantification of OB motility during regeneration, live-imaging time-lapses of bony-rays, including the segment 0 and segment —1, were used. Quantification was performed using a custom Matlab script that performs all the workflow.

Both GFP and brightfield BF channels were gaussian filtered sigma 2 to reduce noise. Intersegment regions were found to define the boundaries between the segments analyzed using the BF channel and a sobel vertical algorithm, dilated with a vertical kernel and small connected components pixels removed resulting in an average line profile for each bony-ray.

Intersegments peaks were found using findpeaks matlab function. OB location was tracked by finding the global GFP intensity center center of mass in segment 0 and —1. GFP line profiles were calculated and summed in height and the intensity center of mass was found in each segment analyzed.

The final result is expressed as a ratio relative OB displacement between the center of mass location and the total segment length 0: Anterior bias; 1: Posterior bias. Number of mitochondria per cell was assessed by quantifying the number of mitochondria and the number of nuclei within the fin, using the Surface tool on IMARIS.

The percentage of mitochondria volume was determined with the same tool, and, for each condition, the volumes of detected mitochondria were grouped into four distinct intervals: smaller than 0. The peaks corresponding to each compound of interest were identified by comparison with standards analysed in the same conditions.

A mass tolerance of 5 ppm and a retention time window tolerance of 10 s were used. Peak areas used for relative quantitation were obtained using the Genesis method. The peak area from AABA was used as an internal quantitation calibrant for the final quantitative data.

Statistical tests, p values, mean and error bars are indicated in the respective figure legends. For sample size see Supplementary file 2d. For OB ArrayXS, fold change was determined based on the normalised data set and expression ratios obtained.

In this data set, a logarithmic base 2 transformation was performed i. log2 expression ratio to make the mapping space symmetric and the up-regulation and down-regulation comparable, prior to the significance test.

Only p-values less than 0. Our editorial process produces two outputs: i public reviews designed to be posted alongside the preprint for the benefit of readers; ii feedback on the manuscript for the authors, including requests for revisions, shown below.

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The authors clearly show that enhanced glycolysis is indispensable for osteoblast dedifferentiation and cell cycle reentry during regeneration; however, they do not provide direct evidence that OXPHOS is diminished. Although treatment with two OXPHOS inhibitors MB6, UK does not inhibit regeneration, the authors provide no rationale or citations for the concentrations of the drugs used here, nor validation that these concentrations are effective in the animal.

Thus, for the authors to maintain their assertion of a "metabolic switch," they should provide further evidence to support the downregulation of OXPHOS e.

Is mitochondria pyruvate carrier downregulated? Are TCA cycle intermediates downregulated? In the absence of such additional support for OXPHOS downregulation, the authors should modify their text title, abstract, and main manuscript and tone down their conclusions accordingly.

Based on the authors' model, enhanced lactate might be predicted to enhance regeneration. The authors are encouraged to repeat this experiment and increase their sample sizes. The figures and text should be modified so that use of these terms is consistent with standard usage. The authors should also comment on why they appear to be detecting proliferation in the distal blastema Figures 6H, S6A-C , in contrast to previous reports.

Positive impact of glycolysis activation or oxidative phosphorylation OXPHOS inhibition. The authors nicely demonstrate the negative impact of glycolysis inhibition on fin regeneration and its cellular mechanisms, such as dedifferentiation, blastema formation and proliferation.

However, the authors did not address another important aspect whether activation of glycolysis or inhibition of OXPHOS can enhance regeneration. One can argue that glycolysis is an essential metabolism and thus inhibition of glycolysis may cause defects of any biological events, including regeneration.

Thus, another important approach is to determine whether metabolic reprogramming can enhance the regenerative program. I understand that the authors tried to address this by targeting OXPHOS but OXPHOS enzymes do not show differential transcription and UK and MB6 do not impact fin regeneration.

To address this further, I recommend the following:. It is unclear whether the authors target to profile only four metabolites with LC-MS analysis Figure 2E. I expect that the authors may be able to get more data from LC-MS, such as a profile of TCA cycle intermediates and key metabolites succinate, citrate, malate, and so on.

Even, there are more glucose metabolites, such as glucosephosphate, fructose 6-phosphate, and so on. As the authors focus on glycolysis and OXPHOS, the profiles of more glycolysis and TCA cycle metabolite changes should be provided.

The authors used two drugs, MB6 and UK However, it is unclear whether treatment of these drugs can efficiently and specifically inhibit OXPHOS. By brief literature search, it is hard to find out papers using these two drugs with animals although there are multiple papers treating these drugs with cells.

Thus, I recommend alternative approaches. Fukuda et al. They used ppargc1a mutants and pdk3b overexpression line, providing evidence that metabolic switch can result in positive effects of heart regeneration.

Magadum et al. Note that pkm2 is a well-characterized glycolytic enzyme in cancer that promotes glucose metabolism toward lactate. Based on Fukuda's work, pkma2 is likely a homolog of pkm2 in zebrafish. The authors consider using one of these animal models to determine whether metabolic reprogramming can enhance fin regeneration.

An alternative approach is to change metabolism by treating drugs. Bae et al. They inhibit the succinate dehydrogenase enzyme complex by treating malonate and Atpenin A5 in mice.

The authors consider treating these drugs and assess whether the metabolic switch can enhance fin regeneration. The blastema is defined as a proliferative cell mass, indicating that there are dedifferentiated cells, and once cells exit blastema they differentiate. In a paper Nechiporuk and Keating, Development , the authors identified there are two distinct populations in the blastema.

Distally located blastema cells are non-proliferative but express blastema marker msxb, naming it as distal blastema DB.

Thereafter, this DB is similarly used by multiple groups in the fin regeneration field as shown by several papers Kang et al. Cell, ; Wehner et al. This DB is a very small domain at the tip of mesenchymal cells. Based on previous works Wehner et al. By contrast, proximal blastema PB contains bilateral zones of proliferative pre-OB runx2 positive cell layers.

As PB is a major blastema region and comprises proliferative cells, the PB ends by emerging differentiated cells, such as osterix positive cells. As patterning zone PZ indicates differentiation, the PZ area starts with osx expressing cells.

This compartmentalization of blastema is standard in fin regeneration. However, the authors define DB, PB, and PZ incorrectly. In Figure 6B, DB actually indicates PB and both PB and PZ indicate PZ.

DB is a very small domain distal to PB, which is not annotated in Figure 6B. Thus, Figure 6 and Sup Figure 5 use incorrect indications and result 5 "Glycolysis suppression leads to ~" is also incorrectly described.

I highly recommend revising the manuscript result section 5 and discussion and figures based on the definition of PB and DB widely used in the field. The authors assess fin regeneration before 48hpa. How about after 48 hours? Are there any bone phenotypes at 4 or 7 dpa or is bone completely lost?

Is there any outcome on OB behavior or fin integrity from blocking glycolysis with 2DG in uninjured fins? However, we highlight that in our data Figure 2C we also observe a clear downregulation of pdha1b , which converts pyruvate into acetyl-CoA, at 6 hpa when changes in energy metabolism start to occur.

The expression profile of the mitochondria pyruvate carriers analysed, mpc1 and mpc2 , was unchanged at 6 and 24 hpa. Given this, we decided to not include these data into the manuscript, but we provide the reviewers the graphs containing the data mentioned above. Unfortunately, for our 6 and 24 hpa samples, the amount of most of the metabolites was very low, bellow the MS instrument sensibility, which limited the data that we could analyse and that we were confident enough to include in the manuscript.

Since this experiment is very time consuming and requires many animals, we could not repeat the assay. We were only able to use the measurements for Citrate, the first metabolic intermediate from TCA cycle generated from acetyl-CoA, and α-KG for the 6 hpa time-point.

Citrate is significantly downregulated, and α-KG remained unchanged, which suggests a lower contribution of acetyl-CoA to fuel TCA cycle and be converted into Citrate, thus potentially reducing of OXPHOS. Given that few TCA intermediates were retrieved from our MS assay, we were not able to further improve our arguments on this point.

We decided to not include these results into the manuscript but provide the data to the reviewers in this document. Mean and SD displayed on the graphs.

This concentration did not lead to mortality nor signs of toxicity in the animals used in our study. This pharmacological compound was previously validated and shown to work in mice for inhibition of mitochondrial OXPHOS Corbet et al.

For MB6, animals were incubated with an already validated concentration, previously described in two studies using zebrafish larvae Dabir et al. In our setting, animals incubated with the same concentration of MB6 did not show signs of toxicity nor increase in mortality.

In addition to the suggested experiments, we also looked at mitochondria dynamics in this context. Usually, changes in cellular metabolism are accompanied and are highly influenced by alterations in the mitochondria morphology, shape and size, due to fusion and fission events or even due to mitochondria biogenesis Wai and Langer, In fact, there are many studies linking mitochondria morphology and function Zemirli et al.

In general terms, while differentiated cells possess a fused and elongated mitochondria network that sustains OXPHOS, the mitochondria of stem cells or proliferating cells, which rely on glycolysis, are smaller and spherical due to increase in fission events Wai and Langer, Here, using a reporter line that labels mitochondria, MLS-GFP, we made two important observations that suggest that changes in metabolism occur in parallel or are accompanied by an increase in fission events at 6 hpa:.

These data suggest that early stages of caudal fin regeneration, at 6 hpa time-point, are characterized by an enhancement of glycolysis that is associated with a potential increase of mitochondria fission. The increase in mitochondrial number and percentage of smaller mitochondria is indicative of mitochondria fission that, in other contexts, is known to be correlated with decrease of mitochondrial OXPHOS activity.

Furthermore, similar observations were shown to occur during zebrafish heart regeneration Honkoop et al. The authors demonstrated that border zone cardiomyocytes near the injury site, were characterized by having smaller and immature mitochondria, suggesting a reduced OXPHOS activity.

Likewise, our data seems to support a tendency to decreased mitochondrial OXPHOS, at least at 6 hpa, however, a more detailed analysis on mitochondrial dynamics using other tools and evaluating other markers related to mitochondria fusion should be done to further support the data.

Thus, we decided to add this data to the manuscript on new Figure 2—figure supplement 2 Lines and in the main text, and Lines ; ; in Material and Methods and expect that these results contribute to clarifying the main message of this work.

We hope the reviewers agree that these modifications make the manuscript more precise and, importantly, are in accordance with the experimental data. Moyes CD, Mathieu-Costello OA, Tsuchiya N, Filburn C, Hansford RG. Mitochondrial biogenesis during cellular differentiation.

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CAS PubMed Google Scholar. Download references. We would like to express our appreciation for the Mayo Clinic Medical Scientist Training Program, the Mayo Clinic Department of Clinical and Translational Science, and the Mayo Clinic Department of Biochemistry and Molecular Biology for fostering an exceptional academic environment.

Josiane Joseph is supported by the National Institute of Health UL1TR, T32GM, and R25GM and the Corella and Bertram Bonner MD, Ph. Mayo Clinic Medical Scientist Training Program, Mayo Clinic, Rochester, MN, USA.

Department of Biochemistry and Molecular Biology, Mayo Clinic, First St SW, Rochester, MN, , USA. You can also search for this author in PubMed Google Scholar. JJ drafted the ideas presented in this work. JD offered suggestions for organization and enhancement of the article.

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Download ePub. Doles ORCID: orcid. Abstract Many chronic disease patients experience a concurrent loss of lean muscle mass. Introduction Skeletal muscle is a vital organ that supports locomotion, respiration, vision, and posture.

Metabolic disruptions in disease that impact skeletal muscle regeneration Skeletal muscle is a highly metabolic tissue and muscle regeneration capacity is dependent on multiple variables. Table 1 Pathologies associated with altered cellular metabolism and satellite cell dysfunction Full size table.

Metabolic interventions that improve skeletal muscle pathology or promote muscle regeneration As highlighted above, various disease processes coincide with dysfunctional skeletal muscle metabolism and impaired satellite cell function.

Select nutritional and small molecule interventions Several common compounds are linked to improved muscle regeneration in diverse disease environments. Hormone replacement Hormonal changes naturally occur with age. Behavioral and hyperbaric oxygen interventions Certain activities, such as weight training, are known to improve muscle mass.

Biologics Mesenchymal stem cell therapies are increasing in popularity and generally regulate inflammatory environments by secreting cytokines. Conclusions In general, satellite cells are often overlooked when evaluating disease associated muscle wasting which may hamper our understanding of this syndrome.

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Thank you for Rejkvenates nature. Rejuvenates metabolism are using a browser version with limited Rejuveenates for CSS. To Rejuvenates metabolism the best Rejuvenates metabolism, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Human mesenchymal stem cells hMSCs promote endogenous tissue regeneration and have become a promising candidate for cell therapy. Among land vertebrates, Rejuvenates metabolism laying Rejuvenates metabolism Body shape fitness out Rejuvenates metabolism to its great reproductive efficiency: producing an mmetabolism daily all year long. Mftabolism production rate makes the laying hen a special model animal to study Rejuveantes general process of Rejuvenates metabolism mrtabolism aging. One unique aspect of hens is their ability to undergo reproductive plasticity and to rejuvenate their reproductive tract during molting, a standard industrial feed restriction protocol for transiently pausing reproduction, followed by improved laying efficiency almost to peak production. Here we use longitudinal metabolomics, immunology, and physiological assays to show that molting promotes reproduction, compresses morbidity, and restores youthfulness when applied to old hens. We identified circulating metabolic biomarkers that quantitatively predict the reproduction and age of individuals. Rejuvenates metabolism


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