Does 18 Hour Fasting Help With Blood Cell Repair?

Cell Stalk Cell. Writer manuscript; available in PMC 2022 Jun v.

Published in final edited form as:

PMCID: PMC4102383

NIHMSID: NIHMS601547

Prolonged Fasting reduces IGF-1/PKA to promote hematopoietic stem cell-based regeneration and opposite immunosuppression

Chia-Wei Cheng,¹ Gregor B. Adams,^two Laura Perin,^three Min Wei,¹ Xiaoying Zhou,² Ben South. Lam,² Stefano Da Sacco,³ Mario Mirisola,⁴ David I. Quinn,^five Tanya B. Dorff,⁵ John J. Kopchick,⁶ and Valter D. Longo^i, ^*

Chia-Wei Cheng

¹Longevity Institute, School of Gerontology, Dept. of Biological Sciences, Academy of Southern California, 3715 McClintock Ave Los Angeles, CA 90089. U.South.

Gregor B. Adams

²Eli and Edythe Broad Heart for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, Academy of Southern California, 1425 San Pablo Street Los Angeles, CA 90033. U.S.

Laura Perin

³Children's Hospital Los Angeles, Division of Urology, Saban Research Found, Academy of Southern California, 4650 Sunset Blvd, Los Angeles, CA 90027, U.S.

Min Wei

¹Longevity Institute, School of Gerontology, Dept. of Biological Sciences, University of Southern California, 3715 McClintock Ave Los Angeles, CA 90089. U.S.

Xiaoying Zhou

^twoEli and Edythe Wide Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, 1425 San Pablo Street Los Angeles, CA 90033. U.South.

Ben Southward. Lam

ⁱⁱEli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, 1425 San Pablo Street Los Angeles, CA 90033. U.S.

Stefano Da Sacco

³Children'south Hospital Los Angeles, Sectionalisation of Urology, Saban Research Institute, University of Southern California, 4650 Dusk Blvd, Los Angeles, CA 90027, U.S.

Mario Mirisola

^ivDepartment of Medical Biotechnology and Forensic, University of Palermo, via Divisi 83, 90133, Palermo, Italy

David I. Quinn

^vTranslational Oncology Program, Kenneth J. Norris Comprehensive Cancer Center, Keck Schoolhouse of Medicine, University of Southern California, 1441 Eastlake Artery Los Angeles, CA 90033, U.S.

Tanya B. Dorff

⁵Translational Oncology Plan, Kenneth J. Norris Comprehensive Cancer Middle, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue Los Angeles, CA 90033, U.S.

John J. Kopchick

^{half dozen}Section of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University Athens, 228 Irvine Hall, Athens, OH 45701, U.S.

Valter D. Longo

¹Longevity Institute, School of Gerontology, Dept. of Biological Sciences, Academy of Southern California, 3715 McClintock Ave Los Angeles, CA 90089. U.S.

SUMMARY

Allowed system defects are at the middle of crumbling and a range of diseases. Here nosotros show that prolonged fasting reduces circulating IGF-1 levels and PKA activity in various cell populations, leading to signal transduction changes in long-term hematopoietic stem cells (LT-HSC) and niche cells that promote stress resistance, self-renewal and lineage-counterbalanced regeneration. Multiple cycles of fasting abated the immunosuppression and mortality caused past chemotherapy, and reversed age-dependent myeloid-bias in mice, in understanding with preliminary data on the protection of lymphocytes from chemotoxicity in fasting patients. The pro-regenerative effects of fasting on stem cells were recapitulated past deficiencies in either IGF-1 or PKA and blunted by exogenous IGF-i. These findings link the reduced levels of IGF-1 caused by fasting, to PKA signaling and establish their crucial function in regulating hematopoietic stem cell protection, self-renewal and regeneration.

INTRODUCTION

Prolonged fasting (PF) lasting 48–120 hours reduces pro-growth signaling and activates pathways that heighten cellular resistance to toxins and stress in mice and humans (Fontana et al., 2010b; Guevara-Aguirre et al., 2022; Holzenberger et al., 2003; Lee and Longo, 2022; Longo et al., 1997). The physiological changes caused by PF are much more than pronounced than those caused by calorie restriction or overnight fast, in part because of the requirement to fully switch to a fat- and ketone bodies-based catabolism after glycogen reserves are depleted during PF (Longo and Mattson, 2022). Studies in mice point that PF can protect them from chemotoxicity by reducing circulating insulinlike growth factor-one (IGF-i) (Lee et al., 2010; Raffaghello et al., 2008). A preliminary example series study also indicates that PF has the potential to better several side furnishings acquired by chemotherapy in humans (Safdie et al., 2009). One of the side effects, myelosuppression, is often dose-limiting in chemotherapy treatment, in role because damage to adult stem/progenitor cells impairs tissue repair and regeneration (Kofman et al., 2022; Mackall et al., 1994; van Tilburg et al., 2022; Williams et al., 2004). Despite the rising interest in food-dependent changes in stem cell populations, piddling is known about how acute or periodic dietary interventions affect the hematopoietic system.

HSPCs residing in the developed bone marrow (BM) are independent inside the Lin⁻Sca-1⁺c-Kit⁺ (LSK) population of cells, which include the self-renewing long-term and curt-term hematopoietic stalk cells (LSK-CD48⁻CD150⁺, LT-HSC and LSK-CD48⁻CD150⁻, ST-HSC) and the multipotent progenitors (LSKCD48⁺, MPP)(Figure S1)(Challen et al., 2009; Rathinam et al., 2022). Together, these cells are responsible for adult hematopoietic regeneration. In the heterogeneous HSCs, several subtypes are identified as Lymphoid-(Ly-HSCs), balanced HSC (Bala-HSC) and Myeloid-HSCs (My-HSCs) according to their singled-out mature blood cell outputs (Figure S1) (Benz et al., 2022; Challen et al., 2010; Muller-Sieburg et al., 2004). In both mice and humans, these HSC subtypes modulate hematopoietic lineage potential and play an important role in lineage-homeostasis during aging (Beerman et al., 2010; Challen et al., 2010; Cho et al., 2008; Pang et al., 2022). Here, we studied the part of multiple PF cycles on chemotherapy–induced and historic period-dependent immunosupression and investigated how PF affects HSC self-renewal, the Ly-, My- and Bala-HSC subtypes as well as their hematopoietic reconstitution outcomes.

RESULTS

Cycles of prolonged fasting (PF) reduce damage in bone marrow stem and progenitor cells and protect mice against chemotoxicity

Chemotherapy drugs cause immunosuppression by inducing Dna harm and cell decease in both peripheral blood (PB) and os marrow (BM), which oft results in long-term impairment of hematopoiesis (Bedford et al., 1984; Yahata et al., 2022). To exam whether PF may protect the hematopoietic organization against immunosuppressive toxicity, mice were fasted or fed an advertizing lib diet (AL) then challenged with cyclophosphamide (CP) for multiple cycles (Figure 1A) (Adams et al., 2007). In agreement with our previous results with etoposide and doxorubicin, we observed a pregnant protective effect of cycles of 48-hours PF against CP-induced mortality (Figure 1B and S1A) (Raffaghello et al., 2008). The PF cycles as well led to a subtract in the DNA damage acquired by CP in leukocytes and BM cells (Figure 1C and S1B).

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Prolonged fasting cycles protect the hematopoietic organization and reverse the chemotherapy-induced hematopoietic suppression

(A) Diagrammatic representation of the experimental procedure to clarify the furnishings of prolonged fasting (PF, 48hr) during 6 cycles of cyclophosphamide chemotherapy (CP, 200mg/kg, i.p.).

(B) Survival curve with vertical dashed lines indicating the pre-chemo starvation period; p<0.01, Log-rank (Mantel-Cox) test; northward=20 (10 male and 10 female).

(C) Deoxyribonucleic acid impairment measurement (olive tail moment) in bone marrow (BM)cells (day 81, 6th recovery phase).

(D) Apoptosis measurement (TUNEL assay) in HSCs and MPP (day 81, 6th recovery phase).

(E) Hematological profile of mice. Total white blood prison cell (WBC), lymphocyte counts and lymphoid/myeloid ratio (L/K) in mice treated with 6 cycles of CP (200mg/kg, i.p.). Each point represents the mean ± south.e.chiliad; horizontal dashed lines signal the ranges of baseline values; * p<0.05, Two-way ANOVA, comparing CP vs. PF+CP during the recovery phase, due north=12 (6 male person and 6 female); Fifty/1000 ratio of peripheral blood (PB) is defined as number of lymphocytes divided by number of myeloid cells (i.due east. granulocytes and monocytes). See also supplementary Effigy S1F and S1G.

(F) Hematological profile of human subjects. Lymphocyte counts and lymphoid/myeloid ratio (L/M) in patients undergoing ii cycles (C1 and C2) of platinum-based doublet chemotherapy in combination with either 24hr or 72hr (48 earlier and 24 hours after chemo) prolonged fasting; D1 and D8 indicate the 1 mean solar day (earlier chemo) and 8 day of each chemotherapy cycle; Each point represents the mean ± s.east.g; ** p<0.01, Two-mode ANOVA; Sample size is indicated in parentheses.

(G) FACS analysis of hematopoietic stalk and progenitor cells (day 84, end of 6^th bike); horizontal dashed lines indicate the baseline value. (H) Proportion of the lymphoid-biased (Ly-HSC), balanced (Bala-HSC) and of the myeloid-biased (My-HSC) hematopoietic stem cells. The markers used are lower side population of LSK (lower-SP^LSK) for My-HSC, middle-SP^LSK for Bala-HSC and upper-SP^LSK for Ly-HSC. The lower panels show a magnification of the SP population in the upper panels.*p<0.05, One-way ANOVA comparing to AL.

(I) BM cells collected from mice treated with either CP or PF+CP were transplanted into the recipient mice. The chimerism of donor-derived cells in PB and that in BM was determined sixteen weeks later on primary BM transplantation. The ratio of lymphocytes to myeloid cells (Fifty/M) in the reconstituted blood was too measured. For (G and I), n= 6 to 10 per group, * p<0.05, ** p<0.01, t-examination comparison the PF with the non-fasted control group both in combination with cyclophosphamide treatment.

To determine whether HSPC protection may be involved in the effects of PF on chemotherapy-induced toxicity, nosotros collected BM cells at the finish of 6 cycles of CP or PF + CP treatments and measured apoptosis. Given that the HSPCs correspond a pocket-size fraction of the total BM, we further examined apoptosis in the subpopulations of these cells (i.e. LT-HSC, ST-HSC and MPP) by performing TUNEL analysis. The results signal that without affecting BM cellularity, PF diminished CP-induced apoptosis in HSPCs (p<0.05, t-test), peculiarly in ST-HSCs and MPPs (Figure 1D, S1C and S1D). The PF-induced protection confronting CP-induced apoptosis was also confirmed by Annexin 5 binding analysis for HSPCs (Effigy S1E).

Prolonged fasting cycles promote lineage-balanced hematopoietic regeneration

To assess whether the protection of HSPCs improved the hematopoietic recovery, we compared the hematological profiles of CP and PF+CP mice at baseline (before CP treatments, after PF), at nadir (2–4 days later on CP) and during the recovery stage (8–10 days later CP) for each wheel of chemotherapy. Multi-cycle CP treatments resulted in a major decline in white blood cell (WBC) counts (Effigy 1E). In the control grouping, WBC suppression, especially the number of lymphocytes, persisted for more than 70 days (6 cycles) (Figure 1E). PF reduced WBC counts independently of chemotherapy and did non prevent the CP-induced decrease in the number of WBCs (Effigy 1E, time 0). However, the beneficial effect of PF was axiomatic starting on cycle 4 (day 39) with the return of lymphocytes to normal levels subsequently the 5^th cycle (day 56) (Figure 1E). At the stop of 6-cycles of treatment, mice in the PF group besides showed normal or close to normal levels of lymphoid cells and normal ratios of lymphoid and myeloid cells (50/M) (Effigy 1E, correct panel). This recovery was observed at similar time-points in iii independent experiments (N=20).

To begin to determine whether PF cycles tin can potentially promote a similar effect in humans, we besides analyzed the hematological profiles of cancer patients from a Stage I clinical trial for the feasibility and safety of a 24–72 hours PF catamenia in combination with chemotherapy. Although 3 dissimilar platinum-based drug combinations were used (Tabular array S1), the results from a Phase I clinical trial indicate that 72 but not 24 hours of PF in combination with chemotherapy were associated with normal lymphocyte counts and maintenance of a normal lineage residual in WBCs (Effigy 1F). These encouraging preliminary results volition need to exist expanded and confirmed in the ongoing Phase II randomized phase of the clinical trial.

In understanding with the effect of PF on the recovery in WBC numbers and improvement in lymphoid/myeloid ratio, results of FACS analyses for stem cell populations indicated an improved preservation of LT-HSCs and ST-HSCs and the enhanced resistance to the myeloid bias in the PF group afterwards half-dozen-cycles of CP handling in mice (Figure 1G and H).

To assess whether the increased HSCs in BM from PF + CP mice can enhance hematopoietic regeneration, we collected BM cells from the CP- or PF+CP-treated mice and transplanted the same number of cells into the immunocompromised (irradiated) recipient mice. Results of this competitive repopulation analysis betoken that, compared to the control group fed ad libtum, the BM cells from mice exposed to six-cycles of CP treatment preceded by PF accept higher regeneration capacity leading to efficient blood reconstitution with improved lymphoid/myeloid ratio (L/M), equally evident from the improved engraftment in blood and in BM (Effigy 1I and S1G and S1H).

Prolonged fasting cycles regulate stalk prison cell populations independently of chemotherapy and assist contrary from immunosenescence

We tested whether the cycles of PF alone could also stimulate HSC self-renewal. Results using BrdU incorporation assays indicated an approximately 6-fold increase of newly generated (BrdU+) HSPCs (i.eastward. LT-HSC, ST-HSC and MPP) in PF mice, which represents 93.7% of the total increment in HSPCs subsequently PF cycles (Effigy 2A). We institute that the increase in LSK cell number is due mainly to an increment in LT-HSCs and ST-HSCs (Figure 2B). By contrast, the number of total BM cells and that of progenitors (i.eastward. MPP, multipotent progenitors; CLP, common lymphoid progenitors; common myeloid progenitor, CMP) was not increased by PF, and, in fact, the number of CMP was slightly decreased during PF (Effigy 2C and S2A).

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Prolonged fasting cycles promote a chemotherapy-contained hematopoietic regeneration

Mice in the control group were fed advertizement libitum and those in the PF group were fasted for one or ii cycles equally indicated. due north=4 to 12 female mice per group.

(A) BrdU incorporation assay for LSK cells. Mice undergoing 24+48hr prolonged fasting were injected (i.p.) with BrdU (0.1mg/grand, twice a day, for 2 days, starting after 24hr of fasting.

(B) Number of long-term hematopoietic stem cells (LT-HSC), brusque-term hematopoietic stalk cells (ST-HSC) and multipotent progenitors (MPP).

(D) Prison cell cycle analysis for BM cells using Ki67 and Hoechst33342.

(Eastward) Apoptosis assay for BM cells using TUNEL analysis.

For (A) to (Due east), * p<0.05, ** p<0.01, *** p<0.005, t-examination comparing the AL-fed controls.

(F) Proportion of the lymphoid-biased (Ly-HSC), balanced (Bala-HSC) and the myeloid-biased (My-HSC) hematopoietic stem cells. The markers used are lower side population of LSK (lower-SP^LSK) for My-HSC, middle-SP^LSK for Bala-HSC and upper-SP^LSK for Ly-HSC.

(M) Number of lymphocytes and myeloid cells in young (6 months, 48hrs fasting) and one-time (eighteen months, 8 cycles of fasting) mice. For (F) and (G), * p<0.05, **p<0.01 and *** p<0.005, one-fashion ANOVA.

Results from prison cell cycle analyses indicate that PF alone induced a major increase of S/G2/One thousand phase in LT-HSCs, ST-HSCs and MPPs (Figure 2D). The pregnant induction in cell wheel entry could explicate at to the lowest degree function of the PF-induced increase in HSCs. In addition to the Ki67/Hoechst33342 staining for cell cycle assay, the PF-induced self-renewal proliferation was also confirmed past analysis using Pyronin Y/Hoechst33342 staining (Effigy S2B). On the other mitt, results from the TUNEL assay indicate that apoptosis was barely detectable in whatsoever subpopulation of HSPCs from either AL-fed or PF mice when no chemotherapy treatment was applied. Apoptosis analysis using Annexin Five and 7AAD indicated similar results (Effigy S2C). Although PF solitary reduces the apoptosis rate in ST-HSCs significantly, the small reduction (from 1.57% to 0.72%) in apoptosis/cellular death could just contribute to a very pocket-sized portion of the PF-induced increase in HSCs and MPP (Figure 2E). However, equally studies of HSCs have shown that induction of proliferation may sometimes be accompanied past an increase of apoptosis (Nakada et al., 2010; Tothova et al., 2007), it is of import to note that this was non observed in PF-induced self-renewal proliferation.

Besides the increase in the number of HSCs and MPP, nosotros besides observed a PF-dependent alteration of lymphoid-, myeloid-biased and counterbalanced-HSCs ratio (Figure 2F, S2D and S2E). Whereas well-nigh HSCs from immature mice are counterbalanced in lymphopoiesis and myelopoiesis, the bulk of HSCs from elderly mice are myeloid biased (Beerman et al., 2010; Challen et al., 2010; Cho et al., 2008; Dykstra et al., 2007; Morita et al., 2010; Muller-Sieburg et al., 2004; Pang et al., 2022). Nosotros therefore investigated if PF cycles can correct this bias in aged mice. Results from eighteen-month erstwhile mice signal that 8 cycles of PF could reverse the age-dependent myeloid bias in HSC subtypes and reverse the effect of crumbling on WBC number in whole blood (Figure 2F and 2G), similar to the changes observed in mice and perhaps patients PF in combination with chemotherapy (Figure 1E,1F and 1H). Taken together, these results suggest that PF cycles tin can too stimulate the HSCs in a chemotherapy-contained fashion which leads to a lineage-counterbalanced hematopoietic regeneration.

Mimicking the effects of prolonged fasting past deficiency in GH/IGF-1 signaling promotes hematopoietic recovery

We previously showed that PF reduces circulating IGF-i levels and that IGF-I deficiency is sufficient to protect mice against chemotherapy toxicity (Lee et al., 2010). To determine if the improved hematopoietic regeneration caused by PF in mice can exist replicated by IGF-1 deficiency, we studied the hematopoietic system in growth hormone receptor knockout (GHRKO) mice, which accept very low circulating and BM IGF-i levels (Al-Regaiey et al., 2005) (Effigy 3A, S3A and Table S2). We institute that CP-induced DNA damage measured by the comet assay in PB and BM cells of GHRKO mice was significantly reduced compared to that in cells from wild-type littermates (Figure 3B). Similar to what was observed in mice undergoing pre-chemo PF cycles, ST-HSCs of the GHRKO mice were protected from CP-induced apoptosis (Figure 3C). Besides, the number of HSCs (i.east. LT-HSCs and ST-HSCs) preserved in the BM of GHRKO mice was higher than that of the wild-type littermates (Figure 3D). An improvement in hematopoietic recovery analogous to that caused by PF, was too observed in GHRKO mice (Effigy 3E).

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Deficiency in GHR/IGF-1 signaling promotes hematopoietic regeneration in both chemo-treated and untreated mice

Measurements were performed in GHRKO and their age matched littermates, with or without treatment with half dozen cycles of CP (200mg/kg, i.p.). n=four to 8 female person mice per group.

(A) BM IGF-1 level in GHRKO mice and PF mice compared to wild type mice fed advertising libitum (WT-AL), * p<0.05, ** p<0.01, one-manner ANOVA.

(B) DNA damage measurement (olive tail moment) in BM cells and mononuclear peripheral claret cells (Lead) from GHRKO and their littermates (WT) (day 81, 6th recovery phase).

(C) Apoptosis measurement (TUNEL assay) in hematopoietic stalk and progenitor cells (twenty-four hour period 81, 6th recovery phase).

(D) Number of hematopoietic stem and progenitor cells (solar day 84, end of vi^th cycle); horizontal dashed lines indicate the chemo-free baseline value.

(East) Total white blood cell (WBC) and lymphocyte counts in PB of GHRKO mice and their littermates (WT); each indicate represents the hateful ± s.e.thou; vertical dashed lines indicate CP treatments; horizontal dashed lines indicate baseline value; * p<0.05, 2-way ANOVA for recovery phases; lymphoid/Myeloid ratio (L/M) afterwards half dozen cycles of CP treatments. Pb L/Thousand ratio is defined equally the number of lymphocytes divided past the number of myeloid cells (i.due east. granulocytes and monocytes). (F) Number of long-term hematopoietic stem cells (LT-HSC) and curt-term hematopoietic stalk cells (ST-HSC). (M) Prison cell bike assay using Ki67 and Hoechst33342.

(H) Number of lymphocytes and myeloid cells in young (age half-dozen months) and old (age 18 months) mice.

For (B to D) and (F to I) *p<0.05, **p<0.01; t-test comparing to the wild-type control.

We found that IGF-ane deficiency also caused the protective effects and the regenerative effects independently of chemotoxicity. Unlike PF mice, GHRKO mice did not have a college frequency of total HSPCs (Effigy S3B). Even so, similarly to what we observed afterwards PF cycles, the frequency of HSCs (i.e. LT-HSCs plus ST-HSCs) was significantly higher in GHRKO mice compared to that in those age and sex matched littermates, with increased cell cycle entry but no detectable differences in apoptosis (Effigy 3F,3G, S3C to S3E). Likewise, the historic period-dependent myeloid bias was non observed in the GHRKO mice (Figure 3H). These data suggest that the periodically reduced IGF-1 signaling caused by PF cycles may play a crucial role in the hematopoietic regeneration observed in PF mice.

Prolonged fasting promote hematopoietic regeneration in IGF-1/PKA dependent mode

To understand the molecular mechanism by which the PF and the GHR/IGF-1 deficiency promote hematopoietic recovery/regeneration, we reanalyzed ii of our previously published microarray data sets and looked for genes whose expression significantly changed in response to PF with a focus on genes similarly affected past exposure of epithelial cells to IGF-1 scarce serum (Guevara-Aguirre et al., 2022; Kim and Volsky, 2005; Kirschner et al., 2009; Lee et al., 2022). In starved mice, the expression of the PKA catalytic subunit alpha (PKACα) was significantly reduced in all tissues tested (Tabular array S3). Similarly, IGF-1 deficient serum from growth hormone receptor-deficient (GHRD) human subjects acquired changes in the expression of both positive and negative regulators of PKA consistent with an inhibition of its kinase activity (Table S4).

As PKA phosphorylates the cAMP response element-binding transcription gene (CREB) at Ser133, p-CREB is ordinarily used as an indicator of intracellular PKA activity (Gonzalez and Montminy, 1989). Using mouse embryonic fibroblasts devoid of the endogenous IGF-1 receptor (R- cells) and those overexpressing the human IGF1R (R+ cell) we showed that CREB phosphorylation is positively regulated by IGF-i/IGF-1R in a PKA-dependent manner, confirming the link between IGF-ane and PKA/CREB signaling in mammalian cells (Figure 4A). IGF-1 receptor (IGF-1R) expression, which was higher in progenitor cells compared to LT-HSCs (Venkatraman et al., 2022), was not afflicted past PF (Figure S4A). Taken together, our in vivo results indicate that PF reduces PKA signaling in BM cells at least in office through reduced IGF-1 levels and PKA action, just without affecting IGF-1R expression (Figure 4B).

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Prolonged fasting promotes IGF-1/PKA dependent hematopoietic regeneration

(A) PKA-dependent phosphorylation of CREB visualized past ICC in mouse embryonic fibroblast (MEFs) devoid of endogenous IGF-1R (R- cells) or overexpressing human IGF1R (R⁺ cells). R+ cells were treated with IGF-1 and compared to cells transfected with PKACα siRNA.

(B) Prolonged fasting (PF) reduces both circulating IGF-1 levels and PKA activeness in BM cells in mice.

IGF-i injection blunted the PF-induced (C) reduction of PKA/pCREB,(D) increase in hematopoietic stem cells, (East-H) The chimerism of donor-derived cells in PB and that in the BM was determined 16 weeks after primary and secondary BM transplantation.. n=4 to viii female mice per group, * p<0.05, **p<0.01 and *** p<0.005, 1-way ANOVA.

To demonstrate that IGF-1 is a mediator of PF-dependent effects on HSCs, we tested whether exogenous IGF-one tin can blunt the effect of PF on HSC number and PKA activity. Fasted mice were injected with IGF-ane (200ug/kg) to reverse the reduction of IGF-one during PF. Results betoken that IGF-1 administration significantly blunted PF-induced reduction of PKA/pCREB in the LSK population, particularly in HSCs (Figure 4C). Information technology also blunted the PF-induced increment in HSCs but not in MPPs (Figure 4D and Table S4). We farther investigated whether the induction in HSCs can lead to enhanced engraftment and whether this effect is IGF-1/PKA-dependent. Results of competitive repopulation assays indicate the PF improved hematopoietic reconstitution in Atomic number 82 and in BM. This effect was blocked past exogenous IGF-1 (Figure 4E, 4F and S4B and S4C). Results of secondary transplantation further confirmed the furnishings in long-term repopulation chapters (Figure 4G and H). Overall, these results strongly support a role for lower IGF-one and the consequent reduced activeness of PKA in PF-dependent stimulation of HSC self-renewal and the comeback in both curt- and long-term hematopoietic repopulation capacities (Effigy 4E to 4H).

As IGF-1R signaling and IGF-1 expression were both reduced in the BM stromal niche cells (Lin⁻CD45⁻) from fasted mice (Figure 5A), we investigated whether the stromal niche could play a part in promoting PF-induced HSC cocky-renewal by reducing IGF-i levels in the microenvironment (as previously shown in Figure 3A). To test this, LT-HSCs were purified (CD45⁺LSK CD150⁺CD48⁻) from mice on either PF or the command diet and and then cantankerous-exposed to the stromal niche cells (CD45⁻Lin⁻ fraction) from mice on either PF or the advert lib diet using co-civilisation systems (Effigy 5B). Notably, LT-HSCs are unable to survive in the absence of niche cells, so the isolated LT-HSCs were not studied alone. Results point that the event of PF on LT-HSC is sufficient to promote the self-renewal of LT-HSC and its capacity to generate ST-HSC and not-LSK progenitors (Lin- not-LSK)(Figure 5C, comparing A to B, C to D). Too, the PF-treated niche cells could increase the generation of ST-HSCs from ad lib diet LT-HSCs (comparison A to C) and increases further the ST-HSC number generated by PF-treated LT-HSCs (comparing B to D). These results confirm the role of LT-HSCs in mediating PF-dependent hematopoietic regeneration but likewise point that niche cells exposed to PF tin can contribute to the ST-HSC component of this regeneration in vitro.

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The role of stromal niche in PF-induced HSC self-renewal

(A) Levels of the indicated proteins in BM stromal niche cells (Lin-CD45-).

(B) Diagrammatic representation of the co-culture experiment.

* p<0.05, **p<0.01 and ***p<0.005, t-test for (A) and one-fashion ANOVA for (C).

Reduction of IGF-i or PKA signaling promotes HSC self-renewal

PKA has conserved pro-aging roles in yeast and mammals (Fabrizio et al., 2001; Rinaldi et al., 2010). In yeast, integration of an extra re-create of the regulatory and inhibitory subunit of PKA, BCY1, (BCY1oe) enhanced whereas mutations in BCY1 that activate PKA decreased, cellular resistance to H₂O₂-induced oxidative stress (Figure 6A) in agreement with our previous results with RAS2- and adenylate cyclase deficient mutants (Fabrizio et al., 2003; Fabrizio et al., 2001). In mammalian cells, information technology was confirmed by u.s.a. and others that disruption of PKA signaling protects against stress (Figure S5A to S5C) (Yan et al., 2007).

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Reduction of IGF-1-PKA signaling promotes hematopoietic stalk cell cocky-renewal

(A) Yeast cells (DBY746 groundwork) overexpressing BCY1 (BCY1oe), which reduces PKA action, or cells carrying mutations that actuate PKA action (bcy1CA1 and bcy1CA2) were grown in SDC for three days and treated with H₂O₂ (50 or 100mM) for 30min at 30°C. Cells were serially diluted and plated onto YPD plates.

(B) PKA-regulated self-renewal pathways in PF mice. The levels of phosphorylation or expression of intracellular proteins in the indicated cellular populations and expression of indicated genes in total BM cells. BM cells were collected from mice with or without 48hr starvation (AL and PF). n=4 female mice per group, **p<0.01, *p<0.05, t-test.

(C) Number of hematopoietic stem cells (per v×ten⁵ total BM) and progenitor cells (LT-HSC, ST-HSC and MPP) under the indicated treatments. * p<0.05, **p<0.01 and *** p<0.005, one-manner ANOVA. See also Effigy S7F and S7G.

BM cells treated with PKA siRNA, IGF-1R siRNA or IGF-one (versus not-treated cells) were transplanted into immuno-compromised recipient mice.

(D) The engraftment in PB was measured at indicated time point after primary transplantation and the engraftment in BM was measured at the cease of the 16 weeks afterward primary transplantation. n=4 to 8 female mice per group, *p<0.05 **p<0.01 and *** p<0.005, one-way ANOVA.

The part of PKA in hematopoietic regeneration, however, is poorly understood. It is known that PKA negatively regulates Foxo1 and positively regulates CREB and G9a (Chen et al., 2008; Gonzalez and Montminy, 1989; Lee et al., 2022; Yamamizu et al., 2022a). FoxOs maintain hematopoietic stress-resistance, self-renewal and lineage homeostasis (Tothova et al., 2007), while CREB and G9a promote hematopoietic lineage-commitment and differentiation (Chen et al., 2022; Yamamizu et al., 2022b). We plant that in PF mice, the reduction of IGF-i/pAkt and PKA/pCREB signaling was associated with an induction of Foxo1 expression and a reduction of G9a (Figure 6B and Figure S5E), but it did not touch on the expressions of Foxo3a and Foxo4 (Figure S5F to S5H). Also, the results indicated that the numbers of ST-HSC and MPP were significantly increased later on handling with PKA siRNA as well as after treatment with IGF-ane siRNA (Figure 6C, Figure S5D and Tabular array S5), in agreement with the finding that inhibition of G9a increases archaic HSCs (Chen et al., 2022).

Given that inhibition of mTOR, another key effector of nutrient signaling, is known to heighten HSC cocky-renewal and maintenance autonomously and not-autonomously, we examined the crosstalk betwixt mTOR and PKA in HSCs and MPPs (Chen et al., 2009; Huang et al., 2022). Ex vivo rapamycin (an mTOR inhibitor) handling alone did not cause an induction in the number of HSCs equally expected based upon previous studies with in vivo treatments (Figure 6C) (Nakada et al., 2010; Yilmaz et al., 2022). This could be due to the need for a longer period of mTor inhibition to achieve HSC induction (Nakada et al., 2010). In fact, when co-treated with PKA siRNA, rapamycin caused an additional induction in ST-HSC and MPP, compared to that caused by PKA-knockdown alone suggesting that diminished PKA signaling promotes the induction of HSCs, which can be further potentiated past mTor inhibition in sure stem and progenitor cell sub-populations (Effigy 6C). Notably, the double inhibition of PKA and mTOR resulted in the synergistic induction in ST-HSC and MPP only blunted the induction in LT-HSC caused by PKA knock-down alone, which is like to what was caused by IGF-R knock-downwards (Figure 6C), and in understanding with the potential role for IGF-1 in the regulation of both PKA and mTOR in HSCs (Fontana et al., 2010a; Longo and Fabrizio, 2002).

The BM cells treated with IGF-1R siRNA or PKA siRNA ex vivo (exBM) were further transplanted into the irradiated recipient mice to examine for their hematopoietic reconstitution capacity. In agreement with the effects observed in PF mice, the PKA or IGF-1R deficient BM cells caused a meaning improvement in engraftment in PB and in BM compared to untreated BM cells (Std)(Figure 6D). The long-term repopulation chapters was also confirmed by secondary transplantation (Figure S5I).

Discussion

When considering changes in gene expression and metabolism, as well equally the levels of various hormones, PF promotes coordinated effects that would be difficult to achieve with any pharmacological or other dietary intervention. In yeast, the key changes responsible for these protective effects of starvation are the down-regulation of the glucose-sensing Ras/adenylate cyclase/PKA and of the amino acid-sensing Tor/Sch9 (S6K) pathways (Effigy 7A)(Fontana et al., 2010b). When mutations in both pathways are combined, cells are extremely resistant to a wide variety of toxins and can live up to 5-fold longer than normal (Fabrizio et al., 2001; Kaeberlein et al., 2005; Kenyon, 2001; Longo and Finch, 2003; Wei et al., 2009). In mammals, mutations that crusade deficiency in the GHR/IGF-i axis promote a range of phenotypes that overlap with those in the highly protected yeast with deficiencies in nutrient signaling pathways including dwarfism, stress resistance, and longevity extension (Effigy 7A)(Lee and Longo, 2022). In fact, cells from GHR/IGF-one scarce mice are protected from multiple forms of stress (Brownish-Borg et al., 2009; Salmon et al., 2005) and the IGF-one deficient (Hat) mice, with an over 70% reduction in circulating IGF-1, are resistant to several chemotherapy drugs (Lee et al., 2010). Here we connect the GHR-IGF-i and the PKA pro-aging pathways past showing that PKA functions downstream of IGF-i to sensitize BM cells in understanding with results in yeast and with the previously established connection betwixt IGF-one and PKA in mammalian neuronal cells (Subramaniam et al., 2005).

An external file that holds a picture, illustration, etc. Object name is nihms601547f7.jpg

PF reduce IGF-1/PKA to promote lineage-balanced hematopoietic regeneration

(A) A simplified model for a partially conserved food signaling PKA pathway in yeast and mammalian cells. Arrows evidence promotion actions and horizontal bars indicate inhibitory actions. GH, growth hormone; AC, adenylate cyclase; PKA, protein kinase A; CREB, cAMP response chemical element-binding protein; Foxo1, Forkhead box protein O1; G9a, H3 Lys-9 methyltransferase.

(B) A simplified model for PF-induced effects on WBC and HSCs. Fasting causes a major reduction in WBCs followed by their replenishment afterwards re-feeding, based on effects on HSCs cocky-renewal resulting in increased progenitor and immune cells. These effects of PF can upshot in reversal of chemotherapy-based immunosuppression simply as well in the rejuvenation of the immune cell profile in onetime mice.

Even so, the studies of growth deficient yeast and mice could non have predicted the remarkable effect of PF cycles in promoting stalk cell-based regeneration of the hematopoietic system. Calorie intake was previously shown to bear on the balance of stem cell self-renewal and differentiation, which is important for somatic maintenance and long-term survival (Bondolfi et al., 2004; Chen et al., 2003; Ertl et al., 2008; Jasper and Jones, 2010; Rafalski and Brunet, 2022; Rando and Chang, 2022). In mice, calorie restriction (CR) promotes the cocky-renewal of intestinal stem cells, muscle stem cell engraftment and neural regeneration, preserves the long-term regenerative capacity of HSC and prevents the decline of HSC number during crumbling in certain mouse strains (Bondolfi et al., 2004; Cerletti et al., 2022; Chen et al., 2003; Ertl et al., 2008; Rafalski and Brunet, 2022; Yilmaz et al., 2022). Reduction of mTOR signaling has been implicated as one of the major molecular mechanisms responsible for the furnishings of CR on enhanced stem jail cell part (Huang et al., 2022; Rafalski and Brunet, 2022; Yilmaz et al., 2022). However, neither CR nor other dietary intervention had previously been shown to promote a coordinated effect leading to the regeneration and/or rejuvenation of a major portion of a system or organ.

Because during PF mammalian organisms minimize free energy expenditure in part by quickly reducing the size of a broad range of tissues, organs, and cellular populations including blood cells, the reversal of this effect during re-feeding represents one of the most potent strategies to regenerate the hematopoietic and possibly other systems and organs in a coordinated mode. Hither nosotros prove that PF causes a major reduction in WBC number, followed, during re-feeding, by a coordinated process able to regenerate this immune system deficiency past changes kickoff during the fasting menses, which include a major increase in LT-HSC and ST-HSC and redirection of the frequency of Ly-HSC/Bala-HSC/My-HSC leading to a lineage-balanced style. In fact, we show that PF lone causes a 28% decrease WBC number, which is fully reversed later on re-feeding (Fig. 7b). Even afterwards WBCs are severely suppressed or damaged equally a consequence of chemotherapy or crumbling, cycles of PF are able to restore the normal WBC number and lineage remainder, suggesting that the organism may be able to exploit its ability to regenerate the hematopoietic arrangement afterward periods of starvation, independently of the cause of the deficiency (Effigy 7B).

In understanding with our results, starvation protects germline stem cells (GSC) and extends reproductive longevity in C. elegans through an adaptive energy shift toward the less committed cells (Angelo and Van Gilst, 2009). In contrast, short-term fasting (≤ 24hr) in Drosophila, promotes the differentiation of hematopoietic progenitors to mature claret cells (Shim et al., 2022). It will be important to determine whether the coordinated regenerative changes observed during PF and re-feeding may resemble at to the lowest degree in part the sophisticated plan responsible for the generation of the hematopoietic arrangement during evolution.

Contempo studies revealed that HSCs rely heavily on the metabolic programs that forestall aerobic metabolism to maintain their quiescent state and self-renewal capacity (Ito et al., 2022; Takubo et al., 2022; Yu et al., 2022). In the example of PF, the energy metabolism is switched progressively from a carbohydrate-based to a fat- and ketone body-based catabolism, which could contribute to HSC self-renewal, in agreement with findings that fat-acid-oxidation promotes HSC asymmetric self-renewal over the symmetric commitment (Ito et al., 2022).

PKA is known to promote lineage specification of HSC through CREB and G9a (Chen et al., 2022; Yamamizu et al., 2022b). Equally inhibition of G9a has been a cardinal strategy to promote reprogramming (Huangfu et al., 2008; Shi et al., 2008), the PF-induced down-regulation of G9a shown here may redirect cell fate through a like process causing the induction in HSCs, analogously to that caused by G9a inhibition (Effigy 5B)(Chen et al., 2022). Contempo studies as well bespeak that PKA tin direct phosphorylate and negatively regulate FoxO1 (Chen et al., 2008; Lee et al., 2022), which has a profound role in stem cell stress resistance, cocky-renewal and pluripotency maintenance (Tothova et al., 2007; Zhang et al., 2022). Whereas PKA is implicated in stem jail cell differentiation, our study suggests that cycles of PF down-regulate IGF-1 and PKA to promote stem cell self-renewal.

A therapeutic claiming of hematopoietic regeneration is to stimulate stem prison cell product for immediate tissue repair while avoiding stem cell depletion under stress (Pang et al., 2022). Our results signal that cycles of an extreme dietary intervention correspond a powerful hateful to modulate key regulators of cellular protection and tissue regeneration only also provide a potential therapy to reverse or alleviate the immunosuppression or immunosenescence caused by chemotherapy handling and aging, respectively, and mayhap by a variety of diseases affecting the hematopoietic system and other systems and organs. The clinical information shown here provide preliminary results supporting the possibility that these effects can besides be translated into effective clinical applications.

EXPERIMENTAL PROCEDURES

Mice

C57BL/6J mice (Jackson laboratory) were used in this study. Mice are either fasted for 48hr or fed ad libitum before chemotherapy treatment. Cyclophosphamide (CP) were administered intraperitoneally (i.p.) at the dose of 200mg/kg every 12–14 days (6 cycles total). IGF-1 was injected (i.p.) at the dose of 100ug/kg, twice a day. vi to 8weeks one-time B6.SJL mice (Taconic) were used every bit recipient mice in the competitive repopulation assay. Genotyping for GHRKO mice was performed every bit shown in Figure S3A. All animal experiments were washed in accordance with the USC Institutional Animate being Care and Apply Commission and NIH guidelines.

Comet analysis

DNA harm (including ssDNA and dsDNA breaks) in freshly nerveless blood and bone marrow (BM) cells was assessed past CometAssay (Trevigen, Inc, Gaithersburg, MD) with a Nikon Eclipse TE300 fluorescent microscope and analyzed with the Comet Score (TriTek Corp., ver1.5). 100–200 cells were scored per experimental sample.

Competitive repopulation assay

BM drove and transplantation were performed equally previously described (Adams et al., 2007). Briefly, BM cells were collected from mice (C57B/6J) treated with 6-cycle CP. 2.5×ten^five BM cells from CP-treated mice were mixed with an equal number of those from a wild-blazon competitor mouse (B6.SJL), and injected into recipient B6.SJL, lethally irradiated 24 h previously with 10 Gy of radiation. The relative contribution of engraftment from the different jail cell sources was assessed by menstruation cytometry of the PB with CD45.2 (C57B/vi) and CD45.1 (B6.SJL) antigens.

FACS analysis

FACS analyses for LT-HSC(LSK-CD48⁻CD150⁺), ST-HSC(LSK-CD48⁻CD150⁻) and MPP(LSK CD48⁺CD150⁻) in BM were performed every bit previously described (Figure S1) (Adams et al., 2007; Challen et al., 2010;). Freshly harvested BM cells were stained with lineage, stalk, and progenitor markers, followed by Annexin-Five/7-AAD staining and TUNEL assay for apoptosis assay or stained with PY/Hoechst33342 or Ki67/Hoechst33342 for cell cycle assay. For competitive repopulation analysis, Lead was nerveless from tail vein. 50–100ul of blood was diluted one:i with PBS and incubated with anti-CD45.1, anti-CD45.ii antibodies and anti-CD11b (BD Biosciences). Analysis was performed with BD FACS diva on LSR II.

BrdU incorporation

For detecting cell genesis, mice were injected (i.p.) with the filter sterilized BrdU 2.0% solution (Sigma) at 0.i mg/grand body weight in PBS, twice a 24-hour interval, for 2 days, starting after 24hr of prolonged fasting (PF mice). BM cells were collected and stained with anti-BrdU combining with the plasma membrane mark antibodies as mentioned above and analyzed on BD FACS diva on an LSR II, according to the manufacturer'southward protocol (BD Biosciences).

Oxidative Stress Assay for yeast

Day three cells were diluted to an OD₆₀₀ of 1 in Grand-phosphate buffer (pH 6) and treated with 50 or 100 mM hydrogen peroxide for xxx min. Serial dilutions of untreated and treated cells were spotted onto YPD plates and incubated at thirty°C for 2–3 days.

Cell culture and treatments

Cell lines and chief cells used in this written report were cultured at 37°C and v% CO₂. Mouse embryonic fibroblast with overexpressed human being IGF1R (R⁺ cells) were derived from IGF1R knockout mice (obtained from Dr. Baserga) and cultured in DMEM/F12 supplemented with 10% FBS. Cells were seeded at eighty% (R⁺ cells) or l% (exBM or hAFSCs) confluence for IGF-1R and PKACα siRNA transfection (100nM, with 1% 10-tremeGENE transfection reagents, Roche) and/or rapamycin treatment (5nM) and the inhibition efficiencies of the of the target proteins was shown as Tabular array SThe IGF-1 induction (10nM, 15min) was performed at 24hr afterward standard incubation. CREB phosphorylation was measured by immunocytochemistry (ICC) with the pCREB-AF488 antibody (jail cell signaling, i:200, overnight at 4°C). Explanted BM cells, isolated HSCs and BM stromal cells were incubated with alpha-MEM+10%FBS. Cell contents were analysis by FACS equally described above.

Statistical analysis

The significance of the differences in mouse survival curves was determined by Log-rank (Mantel-Cox). Unless otherwise indicated in Figure legends, data are presented as means±sem. Student's t-tests for ii groups and ANOVA for multiple groups were used to appraise statistical significance (*p<0.05, **p<0.01, ***p<0.005).

Highlights

Prolonged fasting down-regulates a novel IGF-1/PKA pathway in stem cells
Prolong fasting protects hematopoietic cells from chemotoxicity
Prolonged fasting cycles promote HSC self-renewal to contrary immunosuppression
Inhibition of IGF-i or PKA signaling mimics the effects of prolonged fasting

Supplementary Textile

01

ACKNOWLEDGMENTS

We thank Dr. Lora Barsky (USC Flow Cytometry Core Facility) for assist in the FACS analysis, and Dr. R. Baserga (Thomas Jefferson University) for providing R+ and R- cells. This report was funded in office by NIH/NIA grants AG20642, AG025135 and P01 AG034906. Five.D.L. has equity interest in Fifty-Nutra, a company that develops medical nutrient.

Footnotes

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