Iron deficiency induces maturation-dependent loss of pancreatic β-cells

Domain: neuroscience
Published: 2026-02-16
Editorial outcome: minor_revision

Abstract
Pancreatic β-cells maintain glucose homeostasis by secreting insulin in response to rising blood glucose, a process fueled by mitochondrial ATP production. Iron, a core cofactor in the electron transport chain, is essential for this metabolic coupling. While the cytotoxic effects of iron overload are well known, the role of iron sufficiency during β-cell development remains unclear. Here, we identify a maturation-dependent requirement for iron in mouse and human β-cells. Using chemical chelation and genetic disruption of transferrin receptor (TFRC)-mediated uptake, we show that immature β-cells depend on iron during metabolic transition to functional maturity. Iron restriction at this stage impairs oxidative metabolism and compromises survival. In contrast, mature β-cells remain resilient to iron depletion, revealing a developmental switch in iron dependency. These findings establish iron as a key metabolic cue in β-cell development and suggest strategies to generate fully functional stem cell-derived β-cells for diabetes modeling and cell replacement therapy.

Introduction
Pancreatic β-cells are central to glucose homeostasis, sensing blood glucose levels and secreting insulin accordingly1. To support this function, β-cells rely on dynamic mitochondrial respiration, nearly doubling ATP production under high glucose to meet metabolic demands2. β-cell health – and, by extension, diabetes risk – is determined by nutrient availability, mitochondrial function, cellular stress, and genetic factors3. Despite its central role in these processes, the influence of iron sufficiency on β-cell health remains poorly defined4.

Iron is an essential micronutrient for all mammalian cells5. Its redox versatility, cycling between ferric (Fe³⁺) and ferrous (Fe²⁺) states, is critical for oxidative metabolism and energy production5. Most cellular iron uptake occurs via the transferrin-transferrin receptor 1 (TFRC) axis, which delivers plasma transferrin-bound iron to TFRC-expressing cells (Fig. 1a). High TFRC expression is characteristic of cell types with elevated iron demands, such as erythroid precursors and proliferating cells6,7. Embryonic lethality in TFRC-deficient mice underscores the essentiality of this pathway8. We previously showed that β-cells upregulate TFRC postnatally, reaching levels exceeding those of neighboring endocrine cells9, yet the extent of their functional reliance on this pathway remains unclear.

Multiple lines of evidence suggest that β-cells are acutely sensitive to iron imbalance. In hereditary hemochromatosis, excess iron accumulates in β-cells, driving their loss and leading to “bronze diabetes”10. Clinical interventions such as phlebotomy and chelation improve insulin secretion, sensitivity, and glycemic control in affected individuals11,12. Even modest iron elevations impair β-cell function and increase type 2 diabetes risk4,13. Post-mortem analyses reveal selective iron accumulation in human β-cells, and not α- or δ-cells, following repeated blood transfusions14, mirrored in rat models of iron overload, where β-cells downregulate TFRC as an adaptive response15.

By contrast, the effects of iron deficiency on β-cell biology remain poorly understood, despite iron deficiency being the most common micronutrient deficiency worldwide16. Recent studies implicate unbalanced iron levels in β-cell dysfunction. In Friedreich’s ataxia, mitochondrial iron overload with cytosolic depletion contributes to diabetes risk17. In iron regulatory protein 2 (Irp2)-deficient mice, iron deficiency impairs proinsulin processing and insulin secretion, leading to glucose intolerance18. Yet, most evidence remains correlative, and the underlying mechanisms linking iron availability to β-cell function are unclear.

Here, we define the role of iron metabolism in pancreatic β-cells using chemical and genetic approaches in both mouse and human systems. We identify a critical dependence on TFRC-mediated iron uptake during early β-cell development, coinciding with the metabolic transition to functional maturity. As β-cells mature, their resilience to iron depletion increases, revealing a developmental switch in iron requirement. These findings establish iron sufficiency as a regulator of β-cell maturation, function, and survival, providing insight into the metabolic programming of β-cells and potential targets for diabetes therapy.

Results
β-cells display age-dependent shifts in iron metabolism-related genes
We previously performed bulk RNA sequencing of sorted pancreatic α-, β-, and δ-cells from neonatal (1-week), adult (12-week), and old (18-month) C57BL/6JRj mice19 (Fig. 1b). To assess age-related changes in iron handling, we performed hierarchical clustering based on iron metabolism-related gene expression, revealing five distinct clusters (Fig. 1c). While α- and δ-cells maintained stable expression profiles across ages, β-cells exhibited marked age-dependent transcriptional shifts. The iron importer transferrin receptor 1 (Tfrc) was highly expressed in neonatal and adult β-cells but lowly in old β-cells (Fig. 1c). The iron exporter ferroportin (Slc40a1) was abundant in neonatal β-cells and sharply reduced in later stages (Fig. 1c). Conversely, iron storage molecules ferritin light (Ftl1) and heavy chains (Fth1) showed low neonatal expression but increased progressively with age (Fig. 1c), confirmed by immunostainings (Data in Rebuttal). These findings suggest dynamic, stage-specific regulation of iron metabolism in β-cells during maturation and aging, in contrast to the transcriptional stability observed in α- and δ-cells.

Iron availability drives distinct transcriptional programs in β-cells
To assess how pancreatic islet cells respond to changes in iron levels, we cultured isolated islets from neonatal (1-week) and adult (12-week) C57BL/6JRj mice for 24 h with either the iron chelator deferoxamine (DFO) or the iron supplement ferric citrate (FeCitr) (Fig. 1d). Neither treatment affected the expression of endocrine markers Ins1 (encoding insulin, β-cells), Gcg (encoding glucagon, α-cells), and Sst (encoding somatostatin, δ-cells) in neonatal (Fig. 1e) or adult islets (Fig. 1f). Islet architecture remained intact (Supplementary Fig. 1a), and immunostaining confirmed stable proportions of α-, β-, and δ-cells across conditions (Supplementary Fig. 1b).

Iron chelation robustly induced Tfrc expression in both neonatal and adult islets, while downregulating iron storage genes Ftl1 and Fth1 in neonatal islets (Fig. 1e–g, Supplementary Fig. 1f). Conversely, iron supplementation suppressed Tfrc and induced Ftl1 and Fth1 at both transcript and protein levels (Fig. 1e–g, Supplementary Fig. 1f). The iron exporter Slc40a1/FPN was modestly induced by FeCitr treatment in both age groups (Fig. 1e–f, Supplementary Fig. 1d, f). The abundance of the iron sensor iron regulatory protein 2 (IRP2) was modestly increased during iron chelation and significantly reduced by iron supplementation (Supplementary Fig. 1e, f).

To define β-cell-specific transcriptional responses, we used a yellow fluorescent protein (R26RYFP) reporter system. Islets from Ins1-Cre; R26RYFP mice were exposed to iron modulators, dissociated, and sorted into YFP+ β-cells and YFP- non-β-cells for gene expression profiling (Fig. 1h, i). High recombination efficiency (~100%) was confirmed by co-immunostaining for insulin and YFP (Supplementary Fig. 1g) and by qRT-PCR of marker genes in sorted populations (Fig. 1j). In β-cells, Tfrc expression increased ~3-fold with DFO and decreased ~5-fold with FeCitr (Fig. 1k). Iron storage genes responded similarly across cell types, with Ftl1 and Fth1 downregulated by chelation and induced by supplementation (Fig. 1k). Notably, Slc40a1 expression was selectively reduced in β-cells following chelation (Fig. 1k). Importantly, 24-hour exposure to DFO or FeCitr did not affect islet insulin content (Supplementary Fig. 1h) or glucose-stimulated insulin secretion (Supplementary Fig. 1i, j), and cell viability remained unaffected after short-term iron modulation (Supplementary Fig. 1k).

Loss of TFRC impairs β-cell function and results in diabetes
To investigate the role of TFRC in β-cells in vivo, we used Ins1-Cre; R26RYFP; Tfrclox/lox (β-Tfrc-KO) mice, enabling β-cell-specific disruption of the Tfrc gene from approximately embryonic day 9.520 (Fig. 2a). Immunoblotting of islets from 3-week-old β-Tfrc-KO mice showed a ~50% reduction in TFRC protein relative to controls, accompanied by decreased ferritin and increased IRP2 levels, indicating cellular iron deficiency (Fig. 2b, Supplementary Fig. 2a, b). Immunostaining of pancreatic sections from 7-week-old animals revealed reduced islet size and a marked loss of TFRC+ β-cells in β-Tfrc-KO mice (Fig. 2c). Transferrin uptake was reduced by ~25% in β-cells lacking TFRC, confirming impaired iron acquisition (Fig. 2d) and resulting in lower islet iron content as measured by ICP-MS (Fig. 2e).

β-Tfrc-KO mice developed hyperglycemia beginning at 4 weeks of age (Fig. 2f, Supplementary Fig. 2c), with all animals exceeding the diabetes threshold (11.1 mmol/L) by 7 weeks (Fig. 2g). Circulating ketone levels were elevated, consistent with insulin deficiency and compensatory ketogenesis (Supplementary Fig. 2d), while body weight remained unchanged (Supplementary Fig. 2e). Glucose tolerance tests confirmed progressive metabolic impairment (Fig. 2h, i, Supplementary Fig. 2f–i). At 5 weeks, plasma C-peptide and proinsulin levels were elevated in a compensatory response to hyperglycemia (Fig. 2j, k). The proinsulin-to-C-peptide ratio (PI:C) remained stable, indicating intact proinsulin processing at this stage (Supplementary Fig. 2j). By 7 weeks, both proinsulin and C-peptide secretion declined, consistent with β-cell failure (Fig. 2j, k). Quantification of β-cell function using homeostatic model assessment (HOMA)-β scores and the fasting C-peptide to glucose ratio confirmed severe β-cell dysfunction in β-Tfrc-KO mice at this stage (Fig. 2l, and Supplementary Fig. 2k). Consistently, the insulinogenic index, as determined by the assessment of ΔC-peptide/Δglycemia during glucose tolerance testing, was impaired in β-Tfrc-KO mice at 6 weeks of age (Fig. 2m).

These findings establish TFRC as a critical regulator of β-cell iron homeostasis, with its loss precipitating β-cell dysfunction, impaired glucose regulation, and the onset of diabetes.

TFRC deletion triggers progressive β-cell loss via apoptosis
To determine whether hyperglycemia in β-Tfrc-KO mice arises from impaired insulin production or secretion, we first assessed pancreatic (pro)insulin content. While total pancreas weight was comparable between groups (Supplementary Fig. 3a), β-Tfrc-KO pancreas contained significantly less proinsulin and insulin by 3 weeks, with near-complete depletion by 7 weeks (Fig. 3a, b).

We next evaluated total pancreas and insulin-positive cell volume at postnatal day 1 (P1), 3 weeks, and 7 weeks. Pancreas volume remained stable across genotypes (Supplementary Fig. 3b), but insulin-positive cell volume was markedly reduced in β-Tfrc-KO mice by 7 weeks (Fig. 3c, Supplementary Fig. 3c), with a scarcity of insulin-positive islets (Fig. 3d), a ~3-fold reduction in mean islet size (Supplementary Fig. 3d), and fewer islets per section (Supplementary Fig. 3e).

Islet endocrine composition was preserved until 3 weeks of age, but a selective β-cell loss became evident by 7 weeks (Fig. 3e, f). Importantly, YFP reporter expression remained confined to the residual insulin-positive cells, indicating that β-cells were lost rather than simply silencing insulin expression (Fig. 3g). While total synaptophysin-positive endocrine volume declined in β-Tfrc-KO pancreas (Fig. 3h, and Supplementary Fig. 3f), total glucagon and total somatostatin volumes remained unaffected (Fig. 3i, j, and Supplementary Fig. 3g, h), excluding transdifferentiation as a major contributor.

To determine whether β-cell loss resulted from cell death, we examined apoptosis and DNA damage. TUNEL staining revealed a significant increase in TUNEL+ INS+ cells in β-Tfrc-KO mice, alongside elevated γH2AX expression (Fig. 3k, l, and Supplementary Fig. 3i-j). Furthermore, condensed cytosolic cytochrome C in β-Tfrc-KO β-cells indicated initiation of the intrinsic apoptotic pathway (Supplementary Fig. 3k).

Since iron also supports cell proliferation7, we assessed β-cell replication via Ki67 staining at P1 and 3 weeks but found no genotype-dependent differences (Supplementary Fig. 3l).

Together, these findings suggest that TFRC loss primarily triggers β-cell apoptosis rather than impairing proliferation or inducing β-cell reprogramming, underscoring its essential role in β-cell survival.

TFRC deletion triggers progressive β-cell loss via apoptosis
To investigate the molecular consequences of Tfrc deletion, we performed single-cell RNA sequencing (scRNA-seq) on islets from 3-week-old control and β-Tfrc-KO mice. At later stages, β-Tfrc-KO islets were not recoverable due to extensive β-cell loss. After strict filtering, we analyzed 9040 high-quality cells, classified into major islet cell types using Tabula Muris-derived markers21. Cell type assignments were validated by canonical marker expression (Fig. 4a, b), and relative cell proportions were normalized across genotypes (Supplementary Fig. 4a), revealing comparable β-cell representation (Fig. 4c).

Despite similar clustering, β-Tfrc-KO β-cells exhibited 182 differentially expressed genes – 173 upregulated, 9 downregulated (padj < 0.05, |log2fc| > 0.58), including Ins1 (Fig. 4d). This reduction likely reflects Cre knock-in at the Ins1 locus rather than an iron-dependent effect, as Ins2 levels were unchanged (Supplementary Fig. 4b, c), with co-localization of Cre and Ins1 supporting this interpretation (Supplementary Fig. 4d, e). Notably, we observed significant upregulation of cyclin-dependent kinase inhibitor 1A (Cdkn1a, encoding p21) (Fig. 4d), a canonical p53 target that induces cell cycle arrest or apoptosis under stress22. Immunostaining confirmed increased p21 in β-Tfrc-KO β-cells (Supplementary Fig. 4f, g), implicating p53-mediated stress responses in the β-cell loss. Other upregulated genes included glyceraldehyde-3-phosphate dehydrogenase (Gapdh), which can act as a moonlighting transferrin receptor23, and Calcium Voltage-Gated Channel Subunit Alpha1 C (Cacna1c), an L-type calcium channel involved in non-transferrin-bound iron uptake7,24, suggesting compensatory mechanisms to circumvent TFRC loss (Fig. 4d). Although exons 3-6 of Tfrc were deleted in β-Tfrc-KO β-cells, scRNA-seq revealed apparent upregulation, likely an artifact of 3’-end transcript capture (Fig. 4d). qRT-PCR with exon-specific primers confirmed detection bias due to retained 3’ transcript fragments (Supplementary Fig. 4h).

KEGG pathway analysis of upregulated genes revealed enrichment in HIF-1 signaling, insulin secretion, and glycolysis, indicating a metabolic shift (Fig. 4e). Notably, the enrichment of the “Coronavirus disease” KEGG pathway reflects increased expression of cytoplasmic and mitochondrial ribosomal proteins, which are canonical components of cellular stress responses. Protein network analysis (MCODE)25 uncovered tightly connected clusters related to ribosome function, insulin secretion, secretory vesicles, glycolysis, and p53 signaling (Fig. 4f). Immunostaining confirmed elevated p53 expression in β-Tfrc-KO β-cells (Fig. 4g), and transcriptomic profiling revealed increased expression of Trp53, Cdkn1a, and key mediators of p53-dependent apoptosis, including Bax, Bbc3 (PUMA), Pmaip1 (Noxa), and Perp (Fig. 4h, i). These genes exhibited both higher mean expression (Fig. 4h) and broader distribution across β-cells in the β-Tfrc-KO condition (Fig. 4i), reinforcing activation of a p53-driven apoptotic program.

Collectively, these data indicate that β-cells lacking TFRC undergo a metabolic reprogramming toward glycolysis and activate stress-associated p53 signaling. These adaptations likely reflect compensatory responses to intracellular iron deficiency and contribute to progressive β-cell apoptosis.

TFRC deletion reduces insulin content and impairs mitochondrial respiration in β-cells
Transcriptomic profiling of β-Tfrc-KO β-cells revealed enrichment of genes associated with insulin secretion and glycolysis, prompting us to assess the functional consequences of Tfrc loss on insulin production and mitochondrial metabolism. At 3 weeks of age, insulin content was significantly reduced in β-Tfrc-KO islets (Fig. 5a). Despite this depletion, glucose-stimulated insulin secretion remained intact in static assays (Fig. 5b–d), indicating preserved exocytotic function at this pre-weaning stage, even if insulin reserves declined.

Transmission electron microscopy revealed three prominent ultrastructural abnormalities in β-Tfrc-KO β-cells: (i) a (marked) reduction in mature insulin granules, consistent with reduced hormone content (Fig. 5e, f), (ii) plasma membrane detachment, suggestive of increased cellular fragility under iron deficiency (Fig. 5e), and (iii) mitochondrial alterations, including reduced matrix density and elongated morphology – hallmarks of compromised mitochondrial integrity and function (Fig. 5e). Supporting these findings, E-cadherin staining confirmed plasma membrane detachment presenting as gaps in intercellular contacts (Supplementary Fig. 5a), while immunostaining for the mitochondrial outer membrane marker TOMM20 revealed reduced and fragmented mitochondria in β-Tfrc-KO islets (Supplementary Fig. 5b), indicating altered mitochondrial abundance and morphology.

Given the essential role of iron in mitochondrial respiration, we next assessed islet oxygen consumption. Compared to controls, β-Tfrc-KO islets exhibited altered respiratory profiles (Fig. 5g), with significantly reduced ATP-linked respiration (Fig. 5h), a blunted acute glucose response (Fig. 5i), and diminished maximal respiration capacity (Fig. 5j). Pre- and post-assay imaging confirmed islet integrity and quality, ensuring that the observed differences reflect genuine mitochondrial dysfunction rather than technical artifacts (Supplementary Fig. 5c).

To directly evaluate metabolic flux, we performed 13C6 glucose tracer metabolomics (Fig. 5k). β-Tfrc-KO islets showed increased fractional incorporation of labeled carbon into the glycolytic intermediates D-glyceraldehyde-3-phosphate and pyruvate, with increased relative abundance of the latter (Fig. 5l–n). This was accompanied by significant enrichment of their m+3 isotopologues and elevated m+3 L-lactate (Fig. 5o–q), providing direct evidence for enhanced glycolytic flux and accelerated glucose catabolism bypassing mitochondrial oxidation.

Together, these data demonstrate that β-Tfrc-KO β-cells undergo a metabolic shift toward glycolysis to compensate for impaired mitochondrial respiration. Despite retaining insulin secretion at the pre-weaning stage, β-cells lacking TFRC exhibit depleted insulin stores, compromised mitochondrial bioenergetics, and structural hallmarks of metabolic stress – which likely predispose them to progressive dysfunction.

Iron supplementation prevents diabetes in β-Tfrc-KO mice
To test whether exogenous iron could rescue β-cell loss, we administered iron dextran to β-Tfrc-KO mice at postnatal weeks 1, 3, 5, and 7 (Fig. 6a). At high concentrations, iron saturates transferrin and increases non-transferrin-bound iron, which can be imported via alternative transporters such as DMT1 (Slc11a2) and ZIP14 (Slc39a14) – both expressed in β-cells26. scRNA-seq revealed upregulation of Slc11a2 and Slc39a14 in β-Tfrc-KO β-cells (Fig. 6b), confirmed by qRT-PCR on sorted YFP+ β-cells (Fig. 6c), indicating activation of compensatory iron uptake pathways.

The efficacy of iron supplementation was confirmed by Perls’ staining of liver sections (Fig. 6d). Remarkably, iron supplementation fully prevented diabetes in β-Tfrc-KO mice: iron-treated β-Tfrc-KO mice remained normoglycemic throughout the study, whereas PBS-treated β-Tfrc-KO mice developed severe hyperglycemia and surpassed the diabetic threshold by 7 weeks (Fig. 6e, f). Iron-treated β-Tfrc-KO mice also displayed significantly higher body weight (Fig. 6g) and exhibited HOMA-β scores equivalent to controls at 12 weeks, indicating preserved β-cell function (Fig. 6h).

Pancreas volume did not differ between groups (Fig. 6i), but insulin-positive cell volume was ~7-fold higher in β-Tfrc-KO + iron mice compared to β-Tfrc-KO + PBS controls (Fig. 6j). Both islet size and number per section were preserved with iron supplementation, while severely reduced in β-Tfrc-KO + PBS mice (Fig. 6k, l). Endocrine cell composition remained intact in β-Tfrc-KO + iron mice, while PBS-treated counterparts exhibited selective β-cell loss (Fig. 6m, n). Notably, DMT1 – a transporter implicated in inflammation-induced iron toxicity27 and dynamically regulated during iron acquisition – was upregulated in both PBS- and iron-treated β-Tfrc-KO islets, with the former retaining few β-cells and the latter maintaining normal β-cell numbers (Fig. 6o).

These findings establish iron deficiency – not Tfrc deletion per se – as the proximate cause of β-cell failure in this model. The complete reversal of diabetes by iron supplementation underscores the critical role of iron homeostasis in preserving β-cell survival and metabolic integrity.

TFRC is essential for early but not adult β-cell survival
Bulk RNA sequencing of sorted C57BL/6JRj β-cells revealed a progressive decline in Tfrc expression with age (Fig. 1c). RNA in situ hybridization confirmed this pattern, showing reduced Tfrc transcript abundance in insulin-positive areas over time (Supplementary Fig. 6a-b), suggesting an age-dependent shift in iron import requirements.

To dissect this dependency, we generated an inducible β-cell-specific Tfrc knockout model (Ins1-CreERT; R26RYFP; Tfrclox/lox, β-Tfrc-iKO) allowing temporal control of Tfrc deletion via tamoxifen (TAM) (Fig. 7a).

We first examined the role of Tfrc in adult β-cells. TAM administration at 10 weeks of age induced efficient recombination, confirmed by insulin/YFP co-staining (~91%) and immunoblot showing marked TFRC depletion in β-Tfrc-iKO islets within 2 weeks (Fig. 7b, Supplementary Fig. 6c–e), accompanied by reduced islet iron content (Supplementary Fig. 6f) and lower ferritin expression (Data in Rebuttal). Body weight remained stable across all groups (CTRL+vehicle (VEH), CTRL+TAM, β-Tfrc-iKO + VEH & β-Tfrc-iKO + TAM) (Supplementary Fig. 6g). Strikingly, Tfrc loss from adult β-cells had no significant effect on glucose homeostasis: non-fasting glucose (Fig. 7c), glucose tolerance (Fig. 7d, e, Supplementary Fig. 6h, i; aside from a transient increase in glucose at 30 min. in β-Tfrc-iKO + TAM at 25 weeks), serum C-peptide (Supplementary Fig. 6j), pancreatic insulin-positive cell volume (Fig. 7f, and Supplementary Fig. 6k, l), and islet cell composition (Fig. 7g, Supplementary Fig. 6m) all remained unchanged. These findings indicate that Tfrc is dispensable for adult β-cell function and survival, likely reflecting decreased reliance on iron import with age – supported by the observation that iron supplementation in β-Tfrc-KO mice was discontinued at 7 weeks without impairing glucose homeostasis at 12 weeks (Fig. 6a). This suggests that with maturation β-cells either acquire compensatory iron uptake mechanisms, exhibit lower iron efflux, reduce iron demands, or recycle intracellular iron more efficiently, ultimately reducing their dependence on TFRC-mediated iron uptake.

We next assessed TFRC dependency during early postnatal life by administering TAM at postnatal day 5. TAM modestly reduced body weight (Supplementary Fig. 7a). Neonatal Tfrc loss induced transient hyperglycemia peaking at 6-7 weeks, with partial resolution thereafter (Fig. 7h). Glucose tolerance was significantly impaired and, similarly, partially normalized with age (Fig. 7i, j, and Supplementary Fig. 7b, c). C-peptide failed to rise appropriately in response to glucose (Supplementary Fig. 7d). Insulin-positive cell volume was reduced (Fig. 7k), an effect also partly induced by TAM itself (Supplementary Fig. 7e, f), consistent with our previous work28. TAM decreased islet number per section (Supplementary Fig. 7g), while islet size was selectively affected by neonatal Tfrc loss (Supplementary Fig. 7h). Islet composition was disrupted in neonatal β-Tfrc-iKO, with selective β-cell loss (Fig. 7l, and Supplementary Fig. 7i). Recombination efficiency in neonatal β-Tfrc-iKO β-cells was high at 3 weeks (88.3% YFP+) but progressively declined to 44.4% at 8 weeks and 33.6% at 28 weeks (Fig. 7m, n, Supplementary Fig. 7j), consistent with selective depletion of Tfrc-deficient YFP+ β-cells over time and compensatory expansion of YFP- (non-recombined) β-cells.

Together, these findings reveal a critical window during which β-cells depend on TFRC-mediated iron import for survival and function. This requirement diminishes with age, highlighting a developmental shift in iron metabolism that supports β-cell maturation and long-term maintenance.

TFRC is dispensable for α- and δ-cell survival and function, highlighting a β-cell-specific requirement
To assess whether TFRC is required beyond β-cells in the endocrine pancreas, we generated α-cell-specific (Gcg-Cre; R26RYFP; Tfrclox/lox, α-Tfrc-KO) (Supplementary Fig. 8a) and δ-cell-specific (Sst-Cre; R26RYFP; Tfrclox/lox, δ-Tfrc-KO) knockout mice (Supplementary Fig. 9a). High recombination efficiency was confirmed in both strains by YFP reporter expression (~84% in α-Tfrc-KO, ~71% in δ-Tfrc-KO) (Supplementary Fig. 8b, 9b). Sorted YFP+ α-cells and δ-cells were validated by hormone-specific expression profiles (Supplementary Fig. 8c, d, 9c, d), and Tfrc deletion was confirmed by qRT-PCR and agarose gel electrophoresis (Supplementary Fig. 8e, f, 9e, f).

During four months of follow-up, α-Tfrc-KO mice maintained normal body weight and non-fasting glucose levels compared to controls (Supplementary Fig. 8g, h). Functional assessment of glucagon secretion via insulin-induced hypoglycemia revealed no differences in blood glucose nadir or recovery kinetics (Supplementary Fig. 8i–l). Islet composition and the proportion of glucagon-positive cells were likewise unaffected (Supplementary Fig. 8m, n).

Similarly, Tfrc deletion from δ-cells had no impact on body weight or glycemia (Supplementary Fig. 9g, h). Impaired δ-cell function typically disrupts paracrine regulation of insulin and glucagon secretion, potentially causing hypoglycemia during glucose tolerance tests or impaired recovery from hypoglycemia after insulin challenge. However, δ-Tfrc-KO mice exhibited glucose tolerance and hypoglycemic responses comparable to controls, with similar glucose area under the curve and nadir values (Supplementary Fig. 9i–n). The percentage of somatostatin-positive cells within islets also remained unchanged (Supplementary Fig. 9o, p).

Together, these findings demonstrate that Tfrc is dispensable for α- and δ-cell survival and function, reinforcing its selective and critical role in β-cells within the endocrine pancreas.

Iron deprivation regulates TFRC expression and survival in human β-cells in a developmental stage-dependent manner
To assess whether our findings in mice extend to human β-cells, we examined the effects of iron perturbation across three human model systems: EndoC-βH1 cells, cadaveric donor islets, and induced pluripotent stem cell (iPSC)-derived β-cell aggregates (Fig. 8a).

EndoC-βH1 cells were transfected with siRNAs to silence TFRC and subsequently exposed to DFO and FeCitr. Neither TFRC knockdown nor iron perturbation alone or in combination affected cell viability (Fig. 8b). Iron deprivation upregulated TFRC, while supplementation suppressed it, irrespective of TFRC knockdown (Fig. 8c). Transcript levels of INS, FTH1, and SLC40A1 remained largely unchanged following TFRC knockdown, only FTL increased slightly (Supplementary Fig. 10a–d).

Similarly, DFO or FeCitr exposure of human adult donor islets did not alter islet morphology (Supplementary Fig. 10e) or hormone expression (Fig. 8d). However, DFO nearly doubled TFRC expression and FeCitr significantly upregulated FTL while suppressing TFRC (Fig. 8d). Functional assays confirmed increased transferrin uptake following iron deprivation and reduced uptake after supplementation (Fig. 8e).

We next used human iPSC to model developing β-cells using a 7-stage protocol followed by long-term (LC) culture29,30. Six-day DFO exposure at the endocrine progenitor stage 5 (S5) severely disrupted aggregate morphology and viability, increasing cell death ~20-fold (Fig. 8f, g, and Supplementary Fig. 10f). Sensitivity to iron depletion decreased with maturation: immature stage 7 (S7) and LC iPSC-β-cells aggregates exhibited reduced and delayed cell death following DFO treatment (Fig. 8f, g, Supplementary Fig. 10g, h and j). Consistent with our prior mouse and human models, iron deprivation upregulated TFRC expression in both S7 and LC aggregates (Fig. 8h, and Supplementary Fig. 10i, k).

To further dissect responses in immature β-cells, we performed RNA sequencing on magnetic-activated cell sorting (MACS)-purified S7 β-cells exposed to DFO (with a mean ± s.e.m. composition of 75.4% ± 5.2 INS+, 4.4% ± 1.9 GCG+, 1.7% ± 0.7 SST+, and 3.9% ± 0.1 polyhormonal cells, n=4 independent differentiations). Differential expression analysis identified 38 significantly altered genes (FDR<0.05, |log2fc|>0.58), including upregulation of TFRC, LDHA, SLC11A2, SLC40A1, HK2, SLC2A1, NDRG1, and IGFBP3 (Fig. 8i). Upregulation of iron import (TFRC and SLC11A2) and glycolysis (HK2, LDHA, SLC2A1) genes suggest a metabolic shift from oxidative phosphorylation toward glycolysis under iron-limited conditions (Fig. 8i). Gene set enrichment analysis confirmed increased glycolysis and iron transport activity, along with downregulation of respiratory chain components (Supplementary Fig. 10l).

Finally, mature LC iPSC-β-cell function remained intact under iron deprivation, with preserved insulin content and glucose- and forskolin-stimulated insulin secretion (Fig. 8j-k, Supplementary Fig. 10m-n).

Collectively, these findings mirror our observations in mice, demonstrating that TFRC is consistently upregulated under iron deprivation and that TFRC-dependent iron uptake is critical for the survival of immature human β-cells but becomes dispensable with β-cell maturation.

Methods
Animals
All animal experiments were conducted following the guidelines of the Ethical Committee of the Vrije Universiteit Brussel. Mice were maintained under controlled conditions with a 14/10h light/dark cycle, ambient temperature of 19-25°C, relative humidity of 50-55%, and ad libitum access to food (Safe Diets, Safe #105) and water.

C57BL/6JRj mice (Janvier) were used for the isolation of neonatal (1-week-old) and adult (12-week-old) islets. Ins1-Cre; R26RYFP mice were obtained through a cross of the Ins1-Cre (The Jackson Laboratory, 026801,59) with the R26RYFP strain (The Jackson Laboratory, 006148,60). Ins1-Cre; R26RYFP; Tfrclox/lox (β-Tfrc-KO) mice were generated by crossing Ins1-Cre; R26RYFP with Tfrclox/lox mice (The Jackson Laboratory, 028363,45). An identical breeding strategy was used to generate Ins1-CreERT; R26RYFP; Tfrclox/lox (β-Tfrc-iKO) (The Jackson Laboratory, 024709,61), GcgCre; R26RYFP; Tfrclox/lox (α-Tfrc-KO) (The Jackson Laboratory, 030663,62), and SstCre; R26RYFP; Tfrclox/lox (δ-Tfrc-KO) (The Jackson Laboratory, 028864,63). Control mice are defined as the control littermates (R26RYFP; Tfrclox/lox), except for experiments where sorting based on R26RYFP reporter expression (Ins1-Cre; R26RYFP, Gcg-Cre; R26RYFP, Sst-Cre; R26RYFP) was necessary.

For genotyping, genomic DNA was extracted from mouse ear biopsies and subjected to genotyping using the Mouse Direct PCR Kit (bimake.com, B40015) and primers listed in Supplementary Table 1.

Neonatal β-Tfrc-iKO mice were subcutaneously (sc) injected with tamoxifen (Sigma-Aldrich, T5648) dissolved in corn oil (Sigma-Aldrich, C8267) at postnatal days 5 and 7, receiving a total dose of 2 mg/mL. Adult (10-week-old) β-Tfrc-iKO mice received a total dose of 20 mg/mL spread over five sc injections in two weeks. Experiments were performed in both sexes; sex was not included as a biological variable in the analysis.

Glucose measurements and blood collection
Body weight and blood glucose levels (non-fasting between 8–10 a.m.) were measured once weekly post-weaning using a glucometer (GlucoMen Areo, Menarini Diagnostics). Blood samples were collected, whereby 100 μL of tail vein blood was collected in a tube containing 10 μL ethylenediaminetetraacetic acid (EDTA)/aprotinin (2.2% (w/v)) (Sigma-Aldrich, A6103). Blood samples were centrifuged for 15 min. at 1500g and plasma was collected. C-peptide levels were determined with mouse C-Peptide ELISA (CrystalChem, 90050), according to manufacturer’s guidelines. The homeostatic model assessment (HOMA)-β-score was used to estimate β-cell function.

Intraperitoneal glucose and insulin tolerance tests
For intraperitoneal (ip) glucose tolerance tests, 12-h fasted mice were ip injected with D-glucose (2g/kg body weight) (Sigma-Aldrich, G8270). Blood glucose was measured at 0, 15, 30, 60, 90, and 120 minutes (min.). Blood samples were collected before and at 15 or 30 min. after glucose injection, and plasma C-peptide was measured as described above. For insulin tolerance tests, 6-h fasted mice were ip injected with insulin (0.75 IU Actrapid/kg body weight) (Novo Nordisk), and blood glucose was measured at 0, 15, 30, 60, 90, and 120 min.

Iron dextran administration
For the iron supplementation experiments, control and β-Tfrc-KO mice were ip injected at 1, 3, 5, and 7 weeks of age with either 25 µL phosphate-buffered saline (PBS) (Gibco, 14190-326) or Ferraject 200 (200 mg/mL), corresponding to a 5 mg iron dextran dose per injection as in ref. 42.

Isolation of neonatal and adult mouse pancreatic islets
For islet isolation at 3 weeks of age or older, the main pancreatic duct was injected with 2 mL ice-cold HBSS (Gibco, Thermo Fisher Scientific, 14025-100) containing 0.140 mL CIzyme RI (PELOBiotech, VitaCyte, 005-1030). The pancreas was digested for 17 min. at 37°C in 1 mL HBSS supplemented with 7.5% (w/v) sodium bicarbonate (Sigma-Aldrich, S8761), 0.25% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich, A1470), and 0.1 mg/mL kanamycin (Sigma-Aldrich, 11815032). Afterward, the tube was shaken and 14 mL HBSS, supplemented as described above, was added. After centrifugation and resuspension, the digest was filtered over a 500-micron filter (PluriSelect Life Science, 43-50500-50). The pellet was resuspended in 14 mL Histopaque 1100, the latter prepared by mixing Histopaque 1077 (Sigma-Aldrich, 10771) with Histopaque 1119 (Sigma-Aldrich, 11191) and centrifuged for 20 min. at 328g without brake. The supernatant containing the islets was transferred to a new tube containing 25 mL HBSS, supplemented as described above. After centrifugation, the islets were resuspended in islet culture medium (RPMI 1640 (Thermo Fisher Scientific, 61870-010) supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich, F9665) and 1% (v/v) Penicillin-Streptomycin (Pen/Strep) (Gibco, Thermo Fisher Scientific, 15140122)) and were allowed to recover at 37°C and 5% CO2 for at least 2h before hand picking and subsequent overnight culture. For the islet isolation of neonatal islets, see Supplementary Methods.

Human pancreatic islets
This research complies with all relevant ethical regulations. Islet isolation from human pancreases was performed at Leiden University Medical Center, which has authorization from the Dutch government (BWBR0008974, 261 5191-CZ/IZ/2615195-4152895-1085345-PZ0) for islet transplantation and associated research. Prior to organ retrieval for all human pancreases, informed research consent was obtained from the donors by Eurotransplant, the centralized donation organization in The Netherlands (BWBR008066, Art13). This consent was acquired by an independent organ donation coordinator unaffiliated to the research team. Experiments with human islets were conducted in accordance with the guidelines of and with the approval of the Medical Ethics Committee of the Vrije Universiteit Brussel and Universitair Ziekenhuis Brussel (B1432020000197). Upon arrival in Brussels, human islets were centrifuged and resuspended in CMRL 1066 medium (Corning, 15-110-CV) supplemented with 10% (v/v) FBS, 1% (v/v) Pen/Strep, 1% (v/v) HEPES (Gibco, Thermo Fisher Scientific, 15-630-080), 1% (v/v) Glutamax (Thermo Fisher Scientific, 35050061), and 1.2 mg/mL nicotinamide (Calbiochem, 481907). Human islets were handpicked and cultured in CMRL 1066 medium, supplemented as described above, at 37°C and 5% CO2. Donor characteristics are available in the “Checklist for Reporting Human Islet Preparations Used in Research”, Supplementary Table 2.

Human β-cell line culture
Human insulin-producing EndoC-βH1 cells were cultured as described previously64. For details on RNA interference, see Supplementary Methods.

iPSC culture
Healthy human iPSCs 1.02329, HEL115.665, ESIBIe003-A-1066, and ULBi015.18CTL were cultured in Matrigel-coated plates (Corning, BDAA356231) in E8 medium as described65. For details on differentiation to β-cells and magnetic-activated cell sorting of S7 aggregates, see Supplementary Methods.

Chemical iron modulation
Islets from neonatal and adult C57BL/6JRj mice, adult Ins1-Cre; R26RYFP mice, human islets, and EndoC-βH1 cells were exposed for 24 h to 100 μmol/L DFO (Sigma-Aldrich, D9533) or 500 μmol/L FeCitr (Sigma-Aldrich, F3388). These concentrations were selected based on dose-response assays in mouse islets, where they produced peak expression of Tfrc (for DFO) and Slc40a1 (for FeCitr), without inducing toxicity as assessed by islet morphology. Cultures were maintained at 37°C and 5% CO2 in RPMI 1640 supplemented with 1% (v/v) Pen/Strep for mouse islets, CMRL 1066 supplemented with 1% (v/v) Pen/Strep, 1% (v/v) HEPES, 1% (v/v) GlutaMAX, and 1.2 mg/mL nicotinamide for human islets, and in DMEM (Life Technologies, 31885023) supplemented with 5.5 μg/mL human transferrin (Sigma-Aldrich, T8158), sodium selenite (Sigma-Aldrich, S1382), 2% (v/v) fatty acid-free BSA (Sigma-Aldrich, A8806), 10 mmol/L nicotinamide (Sigma-Aldrich, 72340-1006), 50 μmol/L β-2-mercaptoethanol (Sigma-Aldrich, M3148), and 1% (v/v) Pen/Strep for EndoC-βH1 cells. The culture medium for neonatal mouse islets was supplemented with 1% (w/v) BSA (Sigma-Aldrich, A1470). iPSC aggregates at S5 were exposed to 100 μmol/L DFO in S5 medium65, and aggregates at S7 and after LC were exposed to 100 μmol/L DFO in Ham’s F-10 medium (Gibco, 41550) supplemented with 0.75% fatty acid-free BSA, 0.5% GlutaMAX (Gibco, 35050038), and 1% Pen/Strep (ThermoFisher, 15070063). Medium was renewed every 2 days. Assessment of cell viability and apoptosis, see Supplementary Methods.

Transferrin incorporation assay
After overnight incubation, islets of Ins1-Cre; R26RYFP and β-Tfrc-KO mice, and human islets were serum-starved for 30 min. in either RPMI 1640 (Thermo Fisher Scientific, 61870-010) supplemented with 0.5% (w/v) BSA (Sigma-Aldrich, A1470), 0.1 mg/mL kanamycin (Gibco, 11815-032), and D-glucose (Sigma-Aldrich, G8270) to a final concentration of 20 mmol/L or CMRL 1066 medium (Corning, 15-110-CV) containing 16.6 mmol/L D-glucose. CF-568-conjugated human transferrin (Biotium, 00083) was added to a final concentration of 25 µg/mL. Incubation was during 30 min. at 37°C in the dark. The 488 nm laser and analyzed in the FITC channel. Following transferrin incorporation, CF-568 was excited by a 561 nm laser and analyzed in the PE channel. FlowJo Software 10.8.2 was used to analyze the data.

Mouse islet single-cell dissociation and fluorescence-activated cell sorting
After overnight culture, mouse islets were collected in a 1.5 mL tube, centrifuged (3 min. at 124g) and dissociated into single cells with NeuroCult Dissociation Solution (Stem Cell Technologies, 05715). For fluorescence-activated cell sorting (FACS), cells were washed with RPMI 1640 + 10% (v/v) FBS before filtering over a 5 mL filter cap tube (Corning, Falcon, 352235). For scRNA-seq, cells were washed with ice-cold PBS + 0.04% (w/v) BSA before filtering with a 40-micron Flowmi cell strainer (Sigma, BAH136800040) into a new 1.5 mL tube on ice. Cell number and viability were measured with a NucleoCounter NC-200 using a Via1 cassette (ChemoMetec, 941-0012).

Islet cell sorting into YFP+ and YFP- fractions was performed with the BD FACSMelody Cell Sorter (BD Biosciences). YFP was excited by the 488 nm laser and analyzed in the FITC channel. Following transferrin incorporation, CF-568 was excited by a 561 nm laser and analyzed in the PE channel. FlowJo Software 10.8.2 was used to analyze the data.

Glucose-stimulated insulin secretion
Islets of 3-week-old CTRL and β-Tfrc-KO mice were preincubated in Ham’s F10 medium (custom formulation, Gibco) supplemented with 0.5% (w/v) BSA, 2 mmol/L CaCl2, 100 U/mL Pen/Strep, and 2 mmol/L Glutamax (without glucose, G0) for 1 h at 37°C and 5% CO2. Afterward, islets were transferred to Ham’s F10 medium supplemented with 2 mmol/L glucose (G2) and incubated for 1 h under the same culture conditions. This step was repeated for stimulation with 20 mmol/L glucose (G20). Medium from G2 and G20 wells was collected to measure insulin release, and islets were lysed with lysis buffer and sonication to determine insulin content. Mouse insulin levels were assayed by ELISA (Mercodia, 10-1247-01).

Fifty iPSC-derived LC β-cell aggregates were washed twice with glucose-free Krebs buffer and then preincubated for 90 min. in 500 μL of β Krebs buffer containing 2.8 mmol/L glucose. The aggregates were sequentially incubated for 30 min. intervals in β Krebs buffer with 2.8, 16.7 mmol/L glucose, and 16.7 mmol/L glucose plus 10 μmol/L forskolin (Sigma-Aldrich, F6886). Supernatants were collected and stored at −80°C. Aggregates were lysed by sonication (Bioruptor Plus, 10 sec., 6 cycles) and extracted with acid-ethanol. Insulin secretion and content were assayed by ELISA (Mercodia, 10-1113-10) and normalized to protein content determined by Bradford assay (Bio-Rad Protein Assay, 5000006).

Mouse pancreatic proinsulin and insulin content
The pancreas of 3- and 7-week-old control and β-Tfrc-KO mice was harvested, packed in aluminum foil, snap-frozen in liquid nitrogen, and stored at -80°C. On the day of processing, the pancreas was weighed, transferred to 10 mL 2 mol/L acetic acid, and cut into small pieces before sonication (duty cycle 3, output control 30 for 30 sec. to 1 min. per sample). Samples were incubated for 1 h on ice with periodic vortexing. Subsequently, sonication and incubation were repeated under the same conditions. Samples were centrifuged for 15 min. at 2050g at 4°C. The supernatant was filtered over a 0.2-micron filter using a syringe. Mouse insulin and proinsulin ELISA were performed according to the manufacturer’s guidelines (Mercodia, 10-1247-01 & 10-1232-01).

RNA extraction, reverse transcription, and quantitative PCR
Isolated mouse islets and FACS-sorted cells were collected in 350 μL RLT (Qiagen, 79216) + 1% (v/v) beta-mercaptoethanol (Sigma-Aldrich, M6250) and stored at -20°C. RNA extraction was performed using the RNeasy Micro Kit (Qiagen, 74004). Reverse transcription was performed as in ref. 67. For qRT-PCR, reactions were composed of 2x TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific, 4444557), nuclease-free water, cDNA, and TaqMan Assay detailed in Supplementary Table 3 (Thermo Fisher Scientific). The assay was run on a QuantStudio 12K Flex (Applied Biosystems, Thermo Fisher Scientific). Gene expression levels were normalized against reference genes, as determined by geNorm analysis in qBase+. Reference genes used are specified in the figure legends.

For EndoC-βH1 and iPSC-β-cells, poly(A)+-RNA was isolated using the Dynabeads mRNA DIRECT Purification kit (Invitrogen, 61012). Reverse transcription was done using the Reverse Transcriptase Core Kit (Eurogentec, RT-RTCK-03). Real-time qRT-PCR amplification was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, 1725274) on the CFX Connect instrument (Bio-Rad) and compared to a standard curve. Expression values were normalized to the geometric mean of reference genes β-actin (ACTB) and Vesicle-Associated Membrane Protein-Associated Protein A (VAPA)68. Primers are listed in Supplementary Table 3.

Transcriptome analysis
Next-generation sequencing data of sorted neonatal, adult, and old mouse α-, β-, and δ-cells was obtained from 19.

For scRNA-seq of islet cells of 3-week-old CTRL and β-Tfrc-KO mice, GEMs and scRNA-seq libraries were prepared with the Chromium Next GEM Single Cell 3’ Kit v3.1, Chromium Next GEM Chip G Single Cell Kit, and the Dual Index Kit TT Set A according to the manufacturer’s instructions using the Chromium Controller (10x Genomics). GEM reverse transcription (GEM-RT) was carried out in a C1000 Touch Thermal Cycler (Bio-Rad) at 53°C for 45 min., followed by inactivation at 85°C for 5 min. Cleanup of the GEM-RT product was performed using Dynabeads MyOne SILANE reagent and the SPRIselect Reagent Kit (Beckman Colter). cDNA amplification involved an initial denaturation at 98°C for 3 min., followed by 11 to 12 cycles at 98°C for 15 sec., 63°C for 20 sec., and 72°C for 1 min., with a final extension at 72°C for 1 min. Subsequent steps included enzymatic fragmentation, size selection, and the addition of adapters and sample indices. Sequencing libraries were loaded onto a NovaSeq 6000 using the Novaseq S2 Reagent Kit (Illumina). This was performed in collaboration with BrightCore at VUB (https://www.brightcore.be/). The binary base call (BCL) files were demultiplexed into individual samples using the Cell Ranger mkfastq pipeline (v. 6.0.2). The Cre transgene sequence was integrated into the mouse reference genome. Using RStudio, we removed ambient contamination using SoupX V1.6.169, leveraging canonical marker genes (Ins1, Ins2, Iapp, Gcg, Ppy, and Sst) representative of the major cell types identified during initial clustering for each sample. The following quality control steps were applied: 1) genes expressed in at least three cells and cells expressing a minimum of 200 genes were retained; 2) potential doublets were detected and removed using scDbIFinder v1.12.070; 3) cells with nFeature_RNA<9000 and nCount_RNA<10.000 were retained; 4) cells with >5% mitochondrial gene content were excluded. We thereby obtained high-quality single cells for downstream analysis. Gene expression normalization was performed using SCTransform, which corrects for sequencing depth across cells. In addition, mitochondrial gene expression was regressed out, as it reflects cell stress and state rather than true relevant biological signal. Next, we identified the top 3.000 highly variable genes for principal component analysis (PCA). Data integration was performed using Harmony V1.2.071, based on the top 50 principal components, with sample identity included as a covariate to control for batch effects. The resulting integrated components were used to generate a Uniform Manifold Approximation and Projection (UMAP) embedding. Finally, cell type annotation was performed using scSorter V0.0.272, based on known marker genes from a previous study21. To assess differential gene expression between control and β-Tfrc-KO β-cells, we used the Wilcoxon rank-sum test, and Bonferroni correction was applied to the resulting P-values. For further analysis of β-cells, we extracted all β-cells and generated a new UMAP embedding using the top 12 principal components, selected based on an elbow plot capturing ~80% of the variance.

For bulk RNA sequencing, total RNA from MACS-purified iPSC-β-cells (MACS protocol see Supplementary Methods, obtained from 4 independent differentiations) was isolated using the RNeasy Plus Micro kit (Qiagen, 74034). RNA concentration and integrity were measured using Bioanalyzer RNA 6000 Nano assay (Agilent, 5067-1511) on Agilent 2100 Bioanalyzer. Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs, E7760L) and Novogene NGS Stranded RNA Library Prep Set (Novogene, PT044). All libraries were sequenced on the Illumina NovaSeq 6000 and NovaSeq X plus instrument (Illumina). Paired-end sequencing (2×151 bp) was performed at 200 million reads. Raw read quality was assessed using FastQC (v0.12.1), followed by removing low-quality reads and adapter sequences using fastp (v0.23.2). Filtered reads were then aligned to the GRCh38 primary assembly (GENCODE v46) using Salmon (v1.10.3), achieving an average mapping rate of 90.2 ± 0.9%. Differential gene expression analysis was performed with tximeta (v1.22.1) and DESeq2 (v1.44.0). Wald tests were used to calculate p-values for individual genes, and multiple testing correction was performed using the Benjamini-Hochberg method to control the false discovery rate. Gene set enrichment analysis (GSEA) was carried out using fgsea (v1.33.1) in combination with Reactome pathway obtained from Human MSigDB Collections (v2024.1). The fgsea computes enrichment scores based on gene-level statistics ranked by effect size, with p-values obtained through permutation testing and adjusted for multiple comparisons using the Benjamini-Hochberg method.

Islet iron content by inductively coupled plasma mass spectrometry (ICP-MS)
The concentration of Fe was quantified for 250 isolated islets. Islets were lysed in 400 µL deionized water in Savillex vials to which 2 mL concentrated (14M) ultrapure HNO3 (Thermo Fisher Scientific) was added, after which the Savillex vials were placed on hot plates for 48 hours at 90°C for sample digestion in a class 100 metal-free clean laboratory equipped with two class 10 laminar flow boxes at the AMGC Large Research Group of the VUB. Samples were subsequently evaporated at 75 °C to near-dryness and re-suspended in 2 mL concentrated ultrapure HNO3 after sonication for 15 min. at room temperature. Following a 5-fold dilution of 1 mL of each sample with 3% (v/v) HNO3 and the addition of indium as an internal standard, measurements were performed using an Agilent Technologies 8900 Advanced Applications tandem ICP-MS instrument, and data were first processed using the Masshunter software before further calculations offline. The mass spectrometer was operated in MS/MS mode, and the collision/reaction cell was pressurized with O2 to minimize spectral interferences and improve stability and precision. The nuclide masses 56Fe and 115In were monitored on mass. Quantification took place by external calibration against a calibration curve prepared from multiple single-element stock solutions. Based on repeated measurements of selected reference materials, the analytical precision and accuracy of the procedure outlined above are estimated to be better than 5% and 10 % relative standard deviation (RSD), respectively. The limit of detection for Fe was determined to be 0.013 ng/g, respectively. A technical blank based on the medium was prepared in parallel to the samples and yielded 0.45 ng/g for Fe, which was subtracted from the results before final calculation to concentrations per islet (in pg/islet).

13C glucose tracing
After overnight culture, 180 islets were transferred to glucose-free RPMI 1640 (Thermo Fisher Scientific, 11879020), supplemented with 10% (v/v) FBS, 1% (v/v) Pen/Strep, and 2 mg/mL 13C6 glucose. After 24 h of culture, islets were washed with ice-cold 150 mmol/L NaCl solution and resuspended in 100 µL ice-cold extraction buffer consisting of 80% methanol supplemented with 3 µM each of arginine/glutamate/tryptophan and 1 µM myristic acid. Samples were vortexed and stored at -80°C for at least 24 h. Next, samples were centrifuged for 15 min. at 13.000g and 4°C, and the supernatant was collected for metabolite analysis by mass spectrometry. The remaining cell pellet was retained for protein quantification using the Micro BCA Protein Assay Kit (Thermo Fisher, 23235). For the mass spectrometry analysis, 10 µL of each sample was loaded onto a Dionex UltiMate 3000 LC System (Thermo Scientific Bremen, Germany) equipped with a C-18 column (Acquity UPLC-HSS T3 1.8 µm; 2.1 × 150 mm, Waters) coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) operating in negative ion mode. A step gradient was carried out using solvent A (10 mM TBA and 15 mM acetic acid) and solvent B (100% methanol). The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B until 2 min. post-injection. A linear gradient to 37% B was carried out until 7 min. and increased to 41% until 14 min. Between 14–26 min., the gradient increased to 95% of B and remained at 95% B for 4 min. At 30 min., the gradient returned to 5% B. The chromatography was stopped at 40 min. The flow was kept constant at 0.25 mL/min., and the column was placed at 40°C throughout the analysis. The MS operated in full scan mode (m/z range: [70.0000-1050.0000]) using a spray voltage of 4.80 kV, capillary temperature of 300°C, sheath gas at 40.0, auxiliary gas at 10.0. The AGC target was set at 3.0 E+006 using a resolution of 70000, with a maximum IT fill time of 512 ms. Data collection was performed using the Xcalibur software (Thermo Scientific). The data analyses were performed by integrating the peak areas (El-Maven-Polly-Elucidata). Technical control conditions included islets cultured in ¹²C glucose medium and blank (no islets) samples prepared in both ¹²C- and ¹³C glucose media.

Seahorse metabolic assay
Two days before the assay, the Seahorse XFe96 spheroid microplate (Agilent, 102959-100) was coated with 100 µg/mL poly-D-lysine (Sigma-Aldrich, P7280). After coating, each well was filled with 180 µL Seahorse XF RPMI medium (Agilent, 103576-100) supplemented with 3 mmol/L glucose (Agilent, 103577-100) and 0.1% (v/v) FBS and kept at 37°C until ready for islet loading. The Seahorse XF sensor cartridge was hydrated using Agilent Seahorse XF Hydrobooster. After overnight culture, islets of 3-week-old control and β-Tfrc-KO mice were seeded in a minimal volume of medium at 20 islets per well. The plate was centrifuged for 7 min. at 46g without brake. Next, the plate was incubated for 1 h at 37°C while pictures were acquired with the BioTek Cytation 5 Cell imaging system. Mitochondrial respiration was assessed using the Seahorse XFe96 extracellular flux analyzer (Agilent Technologies) equipped with a thermal tray compatible with the spheroid plate. Basal respiration was measured in 3 mmol/L glucose Seahorse XF RPMI + 0.1% (v/v) FBS medium. Subsequently, islets were exposed to glucose (final concentration 20 mmol/L), followed by 4 µmol/L Oligomycin A, 2 µmol/L FCCP, and 2.5 µmol/L Rotenone/Antimycin A. After the assay, wells were imaged with the BioTek Cytation 5 Cell imaging system to document post-assay islet quality, and only wells in which islets remained stably attached to the plate surface were included in the analysis.

Immunostaining and microscopy
Blood cells were flushed from the circulation of anesthetized mice via systemic perfusion with PBS. The pancreas was harvested and fixed overnight at room temperature in 4% formaldehyde (VWR, VWRK4078.9010), followed by paraffin-embedding (Leica TP1020 Tissue Processor, Leica Biosystems) and sectioning (5 µm). Pancreas sections were dewaxed with xylene and rehydrated with ethanol. Depending on the antigen, tissue sections were subjected to heat- or protease-induced epitope retrieval. Sections were incubated for 30 min. with casein block buffer (Thermo Scientific, 37528), diluted ¼ in PBS + 0.1% (v/v) Tween-20 (PBST) and stained overnight at 4°C with antibodies listed in Supplementary Table 4. After washing with PBST, sections were incubated for 1 h at room temperature with fluorescently labeled secondary antibodies (Supplementary Table 4). Nuclei were stained with 2 µg/mL Hoechst 33342 (Sigma-Aldrich, 14533). After washing (3 × 5 min. PBST), mounting was done with fluoroshield mounting medium (Abcam, ab104135). For TUNEL, immunohistochemical staining, Perls’ staining and RNA in situ hybridization and transmission electron microscopy, see Supplementary Methods.

Fluorescent images were acquired using an Olympus BX61 microscope, while brightfield images were captured using an Aperio GT450 (Leica biosystems).

For iPSC-β-cell aggregates, dissociated cells were seeded in S7 medium containing 10 µmol/L ROCK inhibitor and plated on Matrigel-coated ICC chambers. After 12 h, cells were washed with PBS, fixed in 4% PFA for 20 min., permeabilized with 0.5% Triton-X100 in PBS for 10 min., washed with PBS, and blocked for 10 min. at room temperature using Ultra Cruz Blocking Reagent (Santa Cruz Biotechnology, SC-516214). Cells were incubated overnight at 4°C with primary antibodies diluted in PBST. After 60 min. incubation with secondary antibodies at room temperature, samples were mounted using Vectashield with DAPI (Vector Laboratories, VEC.H-1200). Image quantification was performed using custom CellProfiler 4 pipelines73. Antibodies are listed in Supplementary Table 4. Immunofluorescence pictures were taken on an Axio Imager A1 fluorescence phase contrast microscope (Carl Zeiss, 3521000414) and brightfield pictures were taken on a Primo Vert inverted microscope (Carl Zeiss, 3842003457).

Immunoblot
Islets were collected, rinsed with ice-cold PBS, and resuspended in 50 µL ice-cold RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, P8849 & P5726). Samples were snap-frozen in liquid nitrogen and stored at -80°C. Samples were sonicated (1 min., 30% output, 30% duty cycle) using a Branson 450 Sonifier, followed by centrifugation at 4°C for 20 min. at maximum g. Protein concentration of the supernatant was determined using the DC Protein assay (Bio-Rad, 5000112). Equal amounts of total protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Total protein per lane was quantified using REVERT Total Protein Stain (LI-COR, 926-11015). Membranes were blocked with 5% (w/v) non-fat milk in TBS, followed by overnight incubation at 4°C with primary antibodies (Supplementary Table 4) in TBST (TBS + 0.1% (v/v) Tween-20) + 5% (w/v) BSA). After washing, membranes were incubated with fluorescently labeled secondary antibodies (LI-COR, see Supplementary Table 4) for 1 h at room temperature. Detection was performed using the LI-COR Odyssey Fc Imaging System, and protein abundance was quantified using Image Studio Software (LI-COR). Calculation of the normalized target signal was done via the total protein stain. Unprocessed scans are provided in Source Data and Supplementary Information.

Statistics
Statistical analyses and graphical presentations were performed using GraphPad Prism 10 and RStudio. All compiled data with error bars are expressed as mean ± s.e.m. Statistical tests are provided in the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Van Mulders et al. have identified a critical role for iron in regulating pancreatic beta cell maturation in both mouse and human models. Using pharmacological approaches and several genetic mouse models, they demonstrate that a lack of iron and transferrin receptor (Tfrc) impairs proper beta cell maturation at early postnatal stages, which consequently affects beta cell health and functionality. Importantly, the absence of iron or Tfrc does not impact adult beta cells, indicating a stage-specific requirement for iron during beta cell development. These findings were also confirmed in human models. Overall, the study is well-designed, includes a large number of high-quality data and important findings, is well written, and addresses a significant gap in the field. I have only a few minor comments that the authors may consider before publication.\nComments:\nCould the authors check some of well-known maturation markers in their analysis and discuss how the mechanism presented here is linked with other factors regulating beta cell maturation.\nFigure 1: The impact of iron deficiency or supplementation was studied over a 24-hour period. I was wondering if there are any data from longer incubation times that assess some of the same parameters.\nLine 211: “Thus, these data reveal that β-cells lacking TFRC undergo a metabolic reprogramming toward glycolysis and activate stress-associated p53 signaling.” In case of feasibility, it would strengthen the conclusion if this claim could be supported by additional experimental evidence.\nFigure 5i-j; Some of the mentioned parameters could be quantified.\nExtended Figure 5h: there is a slightly increase in the CKO line. Would be better to mention in the text.\nThe stem cell differentiation data are based on S5 and S7 treatments. Since S5 cells are predominantly endocrine progenitors, treatment at this stage may influence lineage commitment. Is it possible to include data comparing treatment effects on less mature beta cells versus more mature cells within this system?\nWould be better to include the p-value to all graphs.\nFigure 3f could be split into three separate graphs to enhance clarity.\nFigure 5j; Is the observed difference statistically significant?", "reviewer_2": "This study employs chemical chelation and genetic knockout approaches to demonstrate that the iron uptake receptor TFRC is essential for immature β-cell survival and function. The authors systematically delineate the dependency of β-cells on iron during developmental maturation, providing valuable insights into β-cell biology. While the findings are conceptually interesting, several methodological and mechanistic aspects require clarification and additional evidence to strengthen the conclusions.\nSpecific Comments:\n1.Figure 1d: The rationale for selecting the ferric citrate concentration to modulate iron levels in islet cultures lacks justification. Please clarify how this concentration aligns with physiological iron levels in humans (e.g., plasma iron in healthy vs. diabetic individuals) or reference existing literature supporting this dosage.\n2.Figure 2k: HOMA-β is an indirect metric of β-cell function and may not fully capture glucose responsiveness. Direct assessment of β-cell function via glucose-stimulated insulin secretion assays is necessary to substantiate functional changes in TFRC-knockout mice.\n3.Figure 3: While Ki67 data at 3 weeks show no proliferation change, β-cell loss in Ins1-Cre-mediated TFRC knockout (initiated embryonically) could arise from impaired proliferation during embryonic or postnatal stages. Quantitative assessment of β-cell mass and additional proliferation data at earlier developmental stages are required to clarify this.\n4.Figure 4c: The single-cell analysis focuses exclusively on β-cells. To demonstrate β-cell-specific effects and evaluate overall islet compositional changes, UMAP plots depicting all islet cell types from both control and β-Tfrc-KO groups should be included.\n5.Incomplete mechanistic validation: Although scRNA-seq and immunofluorescence indicate p53/CDKN1A pathway activation upon TFRC loss, functional evidence is lacking. Rescue experiments (e.g., p53 or HIF-1α pathway inhibition) are needed to determine whether these pathways are necessary for apoptosis or merely correlative.\n6.Figure 4: Transcriptional changes alone are insufficient to confirm metabolic reprogramming. Functional validation (e.g., Seahorse glycolytic flux assays) is required to support claims of increased glycolytic activity.\n7.Figures 5e–f: Differences in insulin content may confound OCR measurements. Seahorse data must be normalized to total cellular protein content to account for potential variations in cell number/mass.\n8.Figure 6: The dose and frequency of iron dextran supplementation (5 mg/injection) lack experimental or literature-based justification. Provide references for this regimen and include plasma iron levels post-injection to verify efficacy.\n9.The rescue of β-cell defects via iron supplementation in KO mice is compelling. However, clinical context is missing: Does human data link early-life iron deficiency to diabetes risk? Provide association data or discuss existing epidemiological evidence to address translational implications.\n10.Figure 7k: Tamoxifen alone reduces neonatal insulin volume, potentially confounding TFRC knockout phenotypes. Provide structural metrics (e.g., islet number, average size) in neonatal tamoxifen-treated controls to isolate tamoxifen-independent effects.\n11.While neonatal KO shows reduced β-cell iron, adult Tfrc knockout lacks direct evidence of intracellular iron depletion. Measure ferritin expression and labile iron pool (LIP) levels in adult β-cells post-knockout to confirm iron deficiency.\n12.The absence of β-cell defects in adult KO under basal conditions does not preclude vulnerability to metabolic stress. Assess β-cell survival/function in Tfrc-deficient adults exposed to high-fat diet or aging to evaluate iron’s role in stress adaptation.", "reviewer_3": "This study investigates the role of iron deficiency in maturation and function of pancreatic β cells. The authors observed that expression of murine transferrin receptor 1 (Tfrc) is relatively high postnatally but slowly declines with age. Mice with targeted pancreatic β cell-specific disruption of Tfrc developed hyperglycemia and diabetes due to progressive apoptotic death of β cells, following mitochondrial dysfunction and switch to glycolytic energy metabolism. This phenotype was only observed when Tfrc was eliminated during early postnatal development but not in adult mice and could be rescued by parenteral supplementation of excess iron. The authors conclude that iron starvation is detrimental to pancreatic β cells during development but not in the adult stage and provide supportive evidence using a human iPSC β cell aggregate model.\nThe findings are novel and offer significant insights on the role of iron in pancreatic β cell function and pathophysiology. The data are of high technical quality and their interpretation is appropriate. The manuscript would further improve by addressing following issues:\n1) It will be important to assess the iron status of pancreatic β cells in various experimental settings: In constitutive Tfrc knockouts before and after iron supplementation, as well as in temporal Tfrc knockouts in young and adult mice. Some iron markers are shown, but not all of them are specific (for instance Slc40a1 mRNA). A comprehensive assessment of iron status can be done by measuring ferritin by Western (as done in Figures 1g and 2b), ferroportin by Western and/or immunohistochemistry, labile iron by a fluorescent probe, and (if possible) IRP2 expression or IRP activity.\n2) Which is the recombination efficiency for Tfrc ablation in β cells? It appears low compared to that in the YFP reporter mouse model in Fig. 1. Why is there only 50% reduction in Tfrc protein and which are the Tfrc mRNA levels?\n3) In line 200, it is stated that Gapdh “facilitates iron uptake through noncanonical siderophore interactions (ref 23)”. My understanding is that according to work by the authors of the review article in ref. 23, Gapdh may function as a moonlighting transferrin receptor, with much lower affinity for transferrin than Tfrc.\n4) In the KEGG pathway analysis (Fig. 4e), the first hit is “coronavirus disease”. What does this mean?\n5) The data in Fig. 5e-h imply a shit from oxidative phosphorylation to glycolysis. Direct assessment of extracellular acidification rates (ECAR) would strengthen this conclusion.\n6) Is the decrease in mature insulin granules of knockout mice shown in Fig. 5j statistically significant?\n7) Can the phenotype of β cell-specific Tfrc knockout mice be rescued by iron dextran injections starting at later time points (in adult animals)?\n8) In Fig. 6b-c, what does the discrepancy in Slc39a14 expression between the scRNASeq and qPCR data mean?\n9) The iron-dependent induction of DMT1 in Tfrc-deficient β cells (Fig. 6n) is very interesting. DMT1 has been reported to mediate excess iron uptake by these cells (Hansen et al, Cell Metab, 2012; PMID 23000401).\n10) In line 272, how much is “efficient recombination”?\n11) Lines 282-284: Another possibility is that mature β cells have limited iron efflux capabilities due to low ferroportin expression and thus retain more iron.\n12) As noted in the Discussion, the responses of Tfrc following treatment of cells with iron or DFO are expected; therefore, some of these data could be shown in the supplement, if space is limiting.\n13) Lines 366-368: “…iron supplementation rescued β cell loss, indicating that alternative iron uptake pathways can compensate when transferrin-mediated delivery is impaired”. This is very likely true; however, there is significant residual Tfrc expression, which may contribute to the rescue when transferrin becomes fully saturated (following iron dextran injections). “Enhanced Tfrc recycling” (line 403) could also be a factor."}, "author_response": "Point-by-point rebuttal for “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.”\nAnnelore Van Mulders1, Lien Willems1, Sophie Coenen1, Stephanie Bourgeois1, Xiaoyan Yi2, Yue Tong2, Gunter Leuckx1, Yves Heremans1, Julie Pierreux1, Laure Degroote1, Toshiaki Sawatani2, Chiara Vinci2, Gamze Ates3, Ann Massie3, Françoise Carlotti4, Eelco de Koning4, Thomas Mandrup-Poulsen5, Clara Zelinsky6, Steven Goderis6, Bart Ghesquière7, Raphael Scharfmann8, Miriam Cnop2,9,10, Nico De Leu1,11,12,*, Willem Staels1,13,*.\nTable of contents\nReviewer #1 ................................................................................................................................................... 3\n1. Could the authors check some of well-known maturation markers in their analysis and discuss how the mechanism presented here is linked with other factors regulating beta cell maturation. ................................................................. 3\n2. Figure 1: The impact of iron deficiency or supplementation was studied over a 24-hour period. I was wondering if there are any data from longer incubation times assess some of the same parameters. ............................................. 6\n3. Line 211: “Thus, these data reveal that β-cells lacking TFRC undergo a metabolic reprogramming toward glycolysis and activate stress-associated p53 signaling.” In case of feasibility, it would strengthen the conclusion if this claim could be supported by additional experimental evidence. .............................................................................................. 7\n4. Figure 5i-j; Some of the mentioned parameters could be quantified. .......................................................................... 13\n5. Extended Figure 5h: there is a slightly increase in the CKO line. Would be better to mention in the text. ................. 15\n6. The stem cell diUerentiation data are based on S5 and S7 treatments. Since S5 cells are predominantly endocrine progenitors, treatment at this stage may influence lineage commitment. Is it possible to include data comparing treatment eUects on less mature beta cells versus more mature cells within this system? ...................................... 17\n7. Would be better to include the p-value to all graphs. .................................................................................................... 18\n8. Figure 3f could be split into three separate graphs to enhance clarity. ........................................................................ 19\n9. Figure 5j; Is the observed diUerence statistically significant? ....................................................................................... 20\nReviewer #2 ................................................................................................................................................. 21\n1. Figure 1d: The rationale for selecting the ferric citrate concentration to modulate iron levels in islet cultures lacks justification. Please clarify how this concentration aligns with physiological iron levels in humans (e.g., plasma iron in healthy vs. diabetic individuals) or reference existing literature supporting this dosage. ....................................... 21\n2. Figure 2k: HOMA-β is an indirect metric of β-cell function and may not fully capture glucose responsiveness. Direct assessment of β-cell function via glucose-stimulated insulin secretion assays is necessary to substantiate functional changes in TFRC-knockout mice. .................................................................................................................. 22\n3. Figure 3: While Ki67 data at 3 weeks show no proliferation change, β-cell loss in Ins1-Cre-mediated TFRC knockout (initiated embryonically) could arise from impaired proliferation during embryonic or postnatal stages. Quantitative assessment of β-cell mass and additional proliferation data at earlier developmental stages are required to clarify this. 23\n4. Figure 4c: The single-cell analysis focuses exclusively on β-cells. To demonstrate β-cell-specific eUects and evaluate overall islet compositional changes, UMAP plots depicting all islet cell types from both control and β-Tfrc-KO groups should be included. ........................................................................................................................................ 24\n5. Incomplete mechanistic validation: Although scRNA-seq and immunofluorescence indicate p53/CDKN1A pathway activation upon TFRC loss, functional evidence is lacking. Rescue experiments (e.g., p53 or HIF-1α pathway inhibition) are needed to determine whether these pathways are necessary for apoptosis or merely correlative. .. 25\n6. Figure 4: Transcriptional changes alone are insuUicient to confirm metabolic reprogramming. Functional validation (e.g., Seahorse glycolytic flux assays) is required to support claims of increased glycolytic activity ........................ 30\n7. Figures 5e–f: DiUerences in insulin content may confound OCR measurements. Seahorse data must be normalized to total cellular protein content to account for potential variations in cell number/mass. ........................................ 32\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 1\n8. Figure 6: The dose and frequency of iron dextran supplementation (5 mg/injection) lack experimental or literature-based justification. Provide references for this regimen and include plasma iron levels post-injection to verify eUicacy. .............................................................................................................................................................................. 33\n9. The rescue of β-cell defects via iron supplementation in KO mice is compelling. However, clinical context is missing: Does human data link early-life iron deficiency to diabetes risk? Provide association data or discuss existing epidemiological evidence to address translational implications. ................................................................................ 35\n10. Figure 7k: Tamoxifen alone reduces neonatal insulin volume, potentially confounding TFRC knockout phenotypes. Provide structural metrics (e.g., islet number, average size) in neonatal tamoxifen-treated controls to isolate tamoxifen-independent eUects. ...................................................................................................................................... 37\n11. While neonatal KO shows reduced β-cell iron, adult Tfrc knockout lacks direct evidence of intracellular iron depletion. Measure ferritin expression and labile iron pool (LIP) levels in adult β-cells post-knockout to confirm iron deficiency. .......................................................................................................................................................................... 38\n12. The absence of β-cell defects in adult KO under basal conditions does not preclude vulnerability to metabolic stress. Assess β-cell survival/function in Tfrc-deficient adults exposed to high-fat diet or aging to evaluate iron’s role in stress adaptation. ......................................................................................................................................................... 40\nReviewer #3 ................................................................................................................................................. 40\n1. It will be important to assess the iron status of pancreatic β cells in various experimental settings: In constitutive Tfrc knockouts before and after iron supplementation, as well as in temporal Tfrc knockouts in young and adult mice. Some iron markers are shown, but not all of them are specific (for instance Slc40a1 mRNA). A comprehensive assessment of iron status can be done by measuring ferritin by Western (as done in Figures 1g and 2b), ferroportin by Western and/or immunohistochemistry, labile iron by a fluorescent probe, and (if possible) IRP2 expression or IRP activity. ......................................................................................................................................................................... 41\n2. Which is the recombination eUiciency for Tfrc ablation in β cells? It appears low compared to that in the YFP reporter mouse model in Fig. 1. Why is there only 50% reduction in Tfrc protein and which are the Tfrc mRNA levels? ........ 46\n3. In line 200, it is stated that Gapdh “facilitates iron uptake through noncanonical siderophore interactions (ref 23)”. My understanding is that according to work by the authors of the review article in ref. 23, Gapdh may function as a moonlighting transferrin receptor, with much lower aUinity for transferrin than Tfrc. ................................................. 48\n4. In the KEGG pathway analysis (Fig. 4e), the first hit is “coronavirus disease”. What does this mean? ...................... 49\n5. The data in Fig. 5e-h imply a shit from oxidative phosphorylation to glycolysis. Direct assessment of extracellular acidification rates (ECAR) would strengthen this conclusion. ...................................................................................... 50\n6. Is the decrease in mature insulin granules of knockout mice shown in Fig. 5j statistically significant? .................... 52\n7. Can the phenotype of β-cell-specific Tfrc knockout mice be rescued by iron dextran injections starting at later time points (in adult animals)? ................................................................................................................................................. 53\n8. In Fig. 6b-c, what does the discrepancy in Slc39a14 expression between the scRNASeq and qPCR data mean? .. 54\n9. The iron-dependent induction of DMT1 in Tfrc-deficient β cells (Fig. 6n) is very interesting. DMT1 has been reported to mediate excess iron uptake by these cells (Hansen et al, Cell Metab, 2012; PMID 23000401). ............................ 55\n10. In line 272, how much is “eUicient recombination”? ..................................................................................................... 56\n11. Lines 282-284: Another possibility is that mature β cells have limited iron eUlux capabilities due to low ferroportin expression and thus retain more iron. ............................................................................................................................. 57\n12. As noted in the Discussion, the responses of Tfrc following treatment of cells with iron or DFO are expected; therefore, some of these data could be shown in the supplement, if space is limiting. ............................................. 58\n13. Lines 366-368: “…iron supplementation rescued β cell loss, indicating that alternative iron uptake pathways can compensate when transferrin-mediated delivery is impaired”. This is very likely true; however, there is significant residual Tfrc expression, which may contribute to the rescue when transferrin becomes fully saturated (following iron dextran injections). “Enhanced Tfrc recycling” (line 403) could also be a factor. ................................................. 59\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 2\nReviewer #1\nIn this study, Van Mulders et al. have identified a critical role for iron in regulating pancreatic beta\ncell maturation in both mouse and human models. Using pharmacological approaches and\nseveral genetic mouse models, they demonstrate that a lack of iron and transferrin receptor (Tfrc)\nimpairs proper beta cell maturation at early postnatal stages, which consequently affects beta\ncell health and functionality. Importantly, the absence of iron or Tfrc does not impact adult beta\ncells, indicating a stage-specific requirement for iron during beta cell development. These\nfindings were also confirmed in human models. Overall, the study is well-designed, includes a\nlarge number of high-quality data and important findings, is well written, and addresses a\nsignificant gap in the field. I have only a few minor comments that the authors may consider\nbefore publication.\nComments of the reviewer and answers of authors:\n1. Could the authors check some of well-known maturation markers in their analysis and\ndiscuss how the mechanism presented here is linked with other factors regulating beta\ncell maturation.\nWe examined the expression of several β-cell maturation markers by qPCR, single-cell RNA\nsequencing, and immunostaining. Due to a loss of islet integrity beyond 3 weeks of age in β-\nTfrc-KO mice, mRNA abundance analyses are restricted to this early timepoint. To investigate\nlater timepoints, we used immunostainings on fixed pancreases.\nThe expression of canonical mature β-cell identity and transcription factors (Pdx1, Nkx6.1,\nNkx2.2, NeuroD1, Mafa, Pax6, Ucn3), genes involved in functional glucose-stimulated insulin\nsecretion machinery (Slc2a2, Gck, Pcsk1, Pcsk2, G6pc2, Abcc8, Pkm), and secretory granule\nand zinc handling genes (Chga, Chgb, Slc30a8) was, in general, similar in 3-week-old control\nand β-Tfrc-KO β-cells (Rebuttal Figure 1).\nFigure 1: Expression of β-cell maturation markers within the\nβ-cell cluster of 3-week-old control (CTRL) and β-Tfrc-KO Figure 2: qPCR for β-cell markers on 3-week-\nmice, assessed by single-cell RNA sequencing.\nold control (CTRL) and β-Tfrc-KO islets.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 3\nThese findings were corroborated by qPCR, which showed no significant differences in β-cell\nmaturation markers between 3-week-old control and β-Tfrc-KO islets (Rebuttal Figure 2).\nTo assess β-cell identity under iron deficiency-induced damage, we immunostained pancreases\nfrom 5-week-old control and β-Tfrc-KO mice. Despite reduced insulin and fewer β-cells, UCN3,\nNKX6.1, PDX1 and MAFA remained expressed in the β-Tfrc-KO β-cells at levels comparable to\nthose of the controls (Rebuttal figure 3).\nFigure 3: Immunostaining for UCN3, NKX6.1, PDX1, and MAFA on 5-week-old control (CTRL) and β-Tfrc-\nKO pancreas sections (n=3 biological replicates). Scale bar = 100 µm.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 4\nCollectively, these data indicate that established β-cell maturation markers, and thus β-cell\nidentity, are largely preserved in β-Tfrc-KO mice, arguing against cellular dedifferentiation.\nInstead, our findings suggest that iron deficiency impairs β-cell survival by disrupting metabolic\nmaturity, rendering β-cells more vulnerable rather than less differentiated. Interestingly, we\nfound that ferritin (FTH1 & FTL1, responsible for iron storage) expression increases with β-cell\nmaturation (Rebuttal Figure 4). We therefore propose ferritin as a potential marker linking iron\nstatus with β-cell maturation.\nFigure 4: Top panel: Heatmap showing the dynamics of iron metabolism-related gene\nexpression over the β-cell life span, and the static expression of these genes in α- and\nδ-cells. Neonatal mice were 7 days old, adult mice 12 weeks old, and old mice 18\nmonths old. Clusters are generated by the dendrogram cut, dotted horizontal line, and\ncolor code above. Ftl1= Ferritin light chain 1, Fth1 = Ferritin heavy chain 1. Bottom\npanel: Representative immunostaining of ferritin (FTH1/FTL1) in pancreatic islets of\n3-week-old (left), 12-week-old (middle), and 28-week-old (right) wild-type mice. Scale\nbar = 100 µm.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 5\n2. Figure 1: The impact of iron deficiency or supplementation was studied over a 24-hour\nperiod. I was wondering if there are any data from longer incubation times assess some\nof the same parameters.\nWe cultured mouse islets for up to 7 days with deferoxamine (DFO) or ferric citrate (FeCitr).\nBy day 3, control and DFO-treated islets showed compromised morphology, survival, and\nintegrity (including islet clumping and central necrosis) compared to FeCitr-treated islets\n(Rebuttal Figure 5). As control islets deteriorated beyond day 3, we focused our analyses on\nthe 24-hour time point for reliable comparison.\nFigure 5: Brightfield images of CTRL, DFO-treated, and FeCitr-treated mouse islets cultured for 7 days.\nThe same experimental setup was applied to human donor islets, with similarly compromised\nmorphology and survival beyond 24 hours of culture (Rebuttal Figure 6).\nFigure 6: Brightfield images of CTRL, DFO-treated, and FeCitr-treated human donor islets cultured for 8 days.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 6\n3. Line 211: “Thus, these data reveal that β-cells lacking TFRC undergo a metabolic\nreprogramming toward glycolysis and activate stress-associated p53 signaling.” In case\nof feasibility, it would strengthen the conclusion if this claim could be supported by\nadditional experimental evidence.\nWe agree with the reviewer’s comment. Therefore, we performed additional experiments to\ndemonstrate increased glycolytic activity and p53-mediated cell death in β-Tfrc-KO mice.\nHowever, measuring glycolytic activity in pancreatic islets using the Seahorse glycolytic flux\nassay is not straightforward. See below the comment from the application specialist at Agilent\nwe contacted for such experiment:\n“It is diJicult – if not near impossible to measure glycolytic activity in islets using the\nSeahorse. That is because what we measure as glycolytic activity is media acidification\n(since protons generated in glycolysis are expelled from cells to the media together with\nlactate). But as you know islets are mostly comprised of beta-cells (at least in rodent islets\n– human islets are a bit more mixed cell types). Beta-cells do not really produce and\nsecrete lactate since glucose metabolism is tightly linked to mitochondrial ATP production\n– that is how they sense blood glucose. That also means that the majority of acidification\nobserved from islets is actually CO2 from mitochondrial metabolism, and that is also why\nyou will often see that the ECAR profile of a mito stress test will behave very similar to the\nOCR responses, where oligomycin will decrease ECAR (opposite most cells which will\nincrease ECAR upon oligomycin injection). Could this be the reason for why you were\nasked to look at glycolytic activity? If so, there is a perfectly good explanation for that ECAR\nresponse.\nThe best way to determine glycolytic activity is with other types of assays like Tritium\nlabeled glucose or the like.”\nTherefore, we conducted 13C-glucose-based metabolomics on isolated islets from 3-week-\nold β-Tfrc-KO and control mice. The labeling patterns showed an enrichment of glycolytic\nintermediates, with the highest fractional contribution of ¹³C atoms in D-glyceraldehyde 3-\nphosphate, pyruvic acid, and lactic acid β-Tfrc-KO islets. In addition, the m+3 isotopic\ncontribution of ¹³C in these metabolites was significantly elevated in β-Tfrc-KO islets (Rebuttal\nFigure 7). These data are direct evidence for a metabolic shift toward glycolysis in β-Tfrc-KO\nβ-cells.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 7\nFigure 7: a, Overview of 13C-glucose labeling workflow and key metabolites analyzed. b-d, Pie charts of the\n6\nrelative abundance (RA) and fractional contribution (FC, i.e. the total percentage 13C-labeling from 13C-\nglucose) of D-glyceraldehyde, pyruvic acid, and L-lactic acid in islets isolated from control and β-Tfrc-KO\nmice. Pie radii show the RA with concentric circles corresponding from center outwards to 0.25, 0.5, 0.75 and\n1 times the largest abundance. The labeled surface fraction and percentage in the center of each pie reflect\nthe FC. n=4 biologically independent mouse islet preparations per group, analysed by t-test for pRA and\nKruskal-Wallis test for pFC using TraVis Pies. e-g, Corrected 13C isotopologues of D-glyceraldehyde, pyruvic\nacid, and L-lactic acid. Analyzed by two-way ANOVA and Šídák's multiple comparisons test. *: p < 0.05, **: p < 0.01, ****: p < 0.0001.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 8\nSecond, we performed additional experiments to investigate the mechanistic cascade by\nwhich TFRC loss activates p53 signaling and results in β-cell death (Rebuttal figure 8):\na) Increased DNA damage\n• Immunostaining for γH2AX (manuscript Figure 3l & extended Figure 3j):\nimmunostaining and quantitative analysis demonstrate a significant increase in\nγH2AX foci in β-cells of β-Tfrc-KO mice, indicating increased DNA double-strand\nbreaks.\nb) Activation of p53\n• Immunostaining for p53 (new data, Rebuttal figure 9): Immunostaining of pancreas\nsections from 7-week-old mice shows markedly higher nuclear p53 signal in β-Tfrc-\nKO islets compared to controls.\n• scRNA-seq protein network analysis (manuscript Figure 4f): Protein-protein\ninteraction analysis (MCODE) identifies enrichment of p53 signaling pathways in β-\nTfrc-KO β-cells.\nc) Induction of p21 and cell cycle arrest\n• Immunostaining for p21 (manuscript extended Figure 4f-g): Immunostaining and\nquantification reveal increased p21 expression in β-Tfrc-KO β-cells.\n• scRNA-seq DEG analysis (manuscript Figure 4d): The Cdkn1a (encoding p21) is\nsignificantly upregulated in β-Tfrc-KO β-cells.\nd) Upregulation of pro-apoptotic genes\n• scRNA-seq analysis (new data, Rebuttal figure 10): β-Tfrc-KO β-cells exhibit higher\nexpression of canonical pro-apoptotic genes, including Bax, Puma (Bbc3), Noxa\n(Pmaip1), Perp, indicative of activation of the intrinsic apoptotic pathway.\ne) Mitochondrial dysfunction\n• Transmission electron microscopy (manuscript Figure 5e): electron micrographs\nreveal mitochondrial abnormalities, including reduced matrix density and\nelongated morphology, which are features indicative of impaired mitochondrial\nintegrity and function.\n• Seahorse mitochondrial stress test (manuscript Figure 5g-j): β-Tfrc-KO islets\nexhibited altered respiratory profiles, with significantly reduced ATP-linked\nrespiration, blunted acute glucose response, and diminished maximal respiration\ncapacity. These indicate impaired mitochondrial bioenergetics in β-Tfrc-KO islets.\n• Immunostaining for TOMM20 (novel data, Rebuttal figure 11): a marker of the\nmitochondrial outer membrane, shows decreased and fragmented mitochondrial\nstaining in β-Tfrc-KO islets, reflecting altered mitochondrial abundance and\nintegrity.\nf) Cytochrome C release\n• Immunostaining for cytochrome C (new data, Rebuttal figure 12): Confocal imaging\nreveals condensed cytosolic cytochrome C localization in β-Tfrc-KO β-cells,\nconsistent with the initiation of apoptosis.\ng) Caspase activation\n• Immunodetection of cleaved caspase 3 and caspase 9: Cleaved caspase-3 was\ntested using both immunoblotting and immunohistochemistry and was\nundetectable with antibody #9661 (Cell Signaling), even in positive control tissues.\nCaspase-9 immunostaining using ab202068 (Abcam) produced a non-specific\nsignal.\nh) Cell death\n• Immunostaining for TUNEL (manuscript Figure 3k & extended Figure 3i): TUNEL\nstaining revealed a significant increase in TUNEL+INS+ cells in β-Tfrc-KO mice,\ndemonstrating elevated β-cell apoptosis.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 9\nFigure 8: Schematic overview of the p53-regulated cell death pathway.\nFigure 9: Immunostainings for p53 (DAB, brown) and INS (immunofluorescent, green) on 7-week-old control (CTRL)\nand β-Tfrc-KO pancreas sections. n=3 biological replicates (#) per group, and per biological replicate, 4 representative\nislets are shown. Scale bar = 100 µm.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 10\nFigure 10: Average expression and percentage of β-cells expressing Trp53, Cdkn1a and key p53-dependent pro-\napoptotic enectors (Bax, Bbc3, Pmaip1, Perp) in β-cells from 3-week-old control (CTRL) and β-Tfrc-KO mice.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 11\nFigure 11: Immunostaining for TOMM20 and NKX6.1 on 7-week-old control (CTRL) and β-Tfrc-KO pancreas\nsections. Scale bar = 10 µm. Yellow squares indicate the location of magnified insets, which are shown in the\nmiddle and right panel.\nFigure 12: Immunostaining for Cytochrome C and NKX6.1 on 7-week-old control (CTR)L and β-Tfrc-KO pancreas\nsections. Scale bar = 10 µm. Yellow squares indicate the location of magnified insets, which are shown in the\nthird and fourth panel.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 12\n4. Figure 5i-j; Some of the mentioned parameters could be quantified.\nWe agree with the reviewer that quantification of the ultrastructural differences between\ncontrol and Tfrc-KO β-cells would be valuable. To address this, we quantified the reduction in\nmature insulin granule numbers from transmission electron micrographs, which aligned well\nwith the changes in (pro-)insulin content in pancreas lysates (manuscript Figure 3a-b). Other\nultrastructural abnormalities, such as plasma membrane detachment and mitochondrial\nalterations characterized by reduced matrix density and elongated morphology, were not\neasily quantifiable, largely due to the inherently low throughput and limited sampling capacity\nof electron microscopy. For example, plasma membrane detachment was absent in control\nsamples and present in multiple β-Tfrc-KO samples, yet the restricted number of islets that\ncan be examined by TEM introduces substantial sampling bias, making robust quantification\nat the islet level difficult.\nTo mitigate these limitations, we performed additional immunostainings for E-cadherin and\nTOMM20 as markers of membrane and mitochondrial integrity, respectively, and used\nconfocal microscopy to increase the number of cells examined. Consistent with the\nultrastructural findings, E-cadherin expression, which mediates cell–cell adhesion, was\nreduced, and cell membrane detachment was evident as empty spaces between cell borders\nin β-Tfrc-KO islets at 7 weeks of age (Rebuttal Figure 13). Similarly, TOMM20 staining revealed\nmarkedly reduced signal intensity and a more fragmented mitochondrial network (Rebuttal\nFigure 14). Although these confocal data were also challenging to quantify rigorously due to\nthe qualitative nature of the observed defects, the increased sampling depth at least allowed\nus to address the major limitation of TEM and to confirm that these abnormalities are\nconsistently present across a larger number of β-cells.\nTaken together, although we did not perform quantitative analysis of all ultrastructural\nfeatures, the complementary confocal data corroborate and strengthen the conclusions\ndrawn from our electron microscopy observations.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 13\nFigure 13: Immunostaining for E-Cadherin and Hoechst for nuclear staining\non 7-week-old control (CTRL) and β-Tfrc-KO pancreas sections. Scale bar =\n10 µm.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 14\nFigure 14: Immunostaining for TOMM20 and NKX6.1 on 7-week-old control (CTRL) and β-Tfrc-KO pancreas\nsections. Scale bar = 10 µm. Yellow squares indicate the location of magnified insets, which are shown in the\nmiddle and right panel.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 15\n5. Extended Figure 5h: there is a slightly increase in the CKO line. Would be better to\nmention in the text.\nWe agree with the reviewer and have updated the manuscript accordingly.\nRevised:\n“Strikingly, Tfrc loss from adult β-cells had no significant esect on glucose homeostasis: non-\nfasting glucose (Fig. 7c), glucose tolerance (Fig. 7d-e, Extended Fig. 6h-i; aside from a\ntransient increase in glucose at 30 min.in β-Tfrc-iKO+TAM at 25 weeks), serum C-peptide\n(Extended Fig. 6j), pancreatic insulin-positive cell volume (Fig. 7f, Extended Fig. 6k-l), and islet\ncell composition (Fig. 7g, Extended Fig. 6m) all remained unchanged.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 16\n6. The stem cell diYerentiation data are based on S5 and S7 treatments. Since S5 cells are\npredominantly endocrine progenitors, treatment at this stage may influence lineage\ncommitment. Is it possible to include data comparing treatment eYects on less mature\nbeta cells versus more mature cells within this system?\nWe agree that the effects of iron modulation should be investigated at different maturation\nstages. Therefore, the original submission included data of S5 cells, S7 cells, and long-\ncultured (LC) iPSC-derived β-cell aggregates (manuscript Figure 8), the latter being S7 cells\ncultured for an additional month to advance maturation. These datasets enable comparison\nof iron deficiency effects between progenitors (S5), less mature (S7) and more mature (LC) β-\nlike cells, offering a broad perspective on stage-dependent iron requirements.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 17\n7. Would be better to include the p-value to all graphs.\nWe thank the reviewer for this suggestion, which is indeed consistent with the layout used in\nseveral Nature Communications papers. However, in our figures, we found that this format\nreduced overall readability (Rebuttal Figure 15). Nonetheless, we are happy to include the\nactual P-values should the editorial team prefer this presentation.\nFigure 15: Representation of p-values, left panel: shown numerically, and right panel: by symbols.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 18\n8. Figure 3f could be split into three separate graphs to enhance clarity.\nThank you for this suggestion. We adapted our original figure (Rebuttal Figure 16, top left) by\nseparating the data into three individual graphs by age (Rebuttal Figure 16, top right). We also\ngenerated an alternative version split by cell type (Rebuttal Figure 16, middle). However, the\nuse of multiple y-axes and figure legends made the presentation feel overly complex.\nTherefore, we propose an alternative version with a higher data-ink ratio by removal of the\ncommon x-axis (Rebuttal Figure 16, bottom right). We are, of course, happy to further refine\nthe figure in collaboration with the editorial team.\nFigure 16: Layout revisions of islet composition, split by age or cell type.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 19\n9. Figure 5j; Is the observed diYerence statistically significant?\nIn the original comparison, the difference was not statistically significant: an unpaired t-test\ncomparing control (n = 5, mean = 0.0016) and β-Tfrc-KO (n = 8, mean = 0.0013) groups\nyielded a p-value of 0.1077. However, this reflected variability among β-Tfrc-KO cells at 4\nweeks, with some showing clear signs of iron deficiency-induced damage (affected cells),\nwhile others were still intact (unaffected cells). To better capture the heterogeneity at this\ndisease stage, we subdivided the β-Tfrc-KO group in affected and unaffected β-cells\n(based on the criterium of cell membrane detachment) revealing that affected cells\ncontained significantly fewer mature granules than both unaffected and control β-cells\n(Rebuttal Figure 17).\nFigure 17: Left panel: Representative electron micrographs of 4-week-old control (CTRL) and β-Tfrc-KO islets, with\nβ-Tfrc-KO islets further divided into unanected and anected β-cells. Yellow arrows indicate cell membrane\ndetachment and mitochondrial abnormalities in β-Tfrc-KO islets. Upper panel: scale bar = 5 µm. Lower panel:\nscale bar = 1 µm. Right panel: Quantification of the number of mature insulin granules per cytosol area, analyzed by one-\nway ANOVA and Tukey's multiple comparisons test.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 20\nReviewer #2\nThis study employs chemical chelation and genetic knockout approaches to demonstrate that the\niron uptake receptor TFRC is essential for immature β-cell survival and function. The authors\nsystematically delineate the dependency of β-cells on iron during developmental maturation,\nproviding valuable insights into β-cell biology. While the findings are conceptually interesting,\nseveral methodological and mechanistic aspects require clarification and additional evidence to\nstrengthen the conclusions.\nComments of the reviewer and answers of authors:\n2. Figure 1d: The rationale for selecting the ferric citrate concentration to modulate iron\nlevels in islet cultures lacks justification. Please clarify how this concentration aligns\nwith physiological iron levels in humans (e.g., plasma iron in healthy vs. diabetic\nindividuals) or reference existing literature supporting this dosage.\nThe ferric citrate concentration (FeCitr, 500 μM) was chosen based on previous studies using\n100-1000 µM to modulate intracellular iron and activate iron-responsive genes such as\nSlc40a1 and Fth1(PMID: 22682226, PMID: 32675293) and further validated by in-house\ndose-response assays. Although physiological plasma iron levels in humans range from 10-\n30 µM (primarily transferrin-bound), higher concentrations are needed in vitro to osset\nreduced bioavailability due to complexation and protein binding to FBS. At 500 µM, FeCitr\nreliably increased Slc40a1 expression without inducing cytotoxicity, indicating a successful\ncellular response to iron excess without overload. This makes it a suitable concentration for\nstudying iron-dependent responses in isolated islets (Rebuttal Figure 18).\nWe now included the rationale for FeCitr concentration in the Materials and Methods\nsection.\nRevised:\n“Islets from neonatal and adult C57BL/6JRj mice, adult Ins1-Cre;R26RYFP mice, human\nislets, and EndoC-βH1 cells were exposed for 24 hours to 100 μmol/L DFO (Sigma-Aldrich,\nD9533) or 500 μmol/L FeCitr (Sigma-Aldrich, F3388). These concentrations were selected\nbased on dose-response assays in mouse islets, where they produced peak expression of\nTfrc (for DFO) and Slc40a1 (for FeCitr), without inducing toxicity as assessed by islet\nmorphology.”\nFigure 18: Dose-dependent induction of Slc40a1 expression in mouse pancreatic islets by ferric\ncitrate (FeCitr). Analyzed by one-way ANOVA and Holm-Šídák's multiple comparisons test. p = 0.0051\ncompared to 0 µM.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 21\n3. Figure 2k: HOMA-β is an indirect metric of β-cell function and may not fully capture\nglucose responsiveness. Direct assessment of β-cell function via glucose-stimulated\ninsulin secretion assays is necessary to substantiate functional changes in TFRC-\nknockout mice.\nTo directly assess β-cell function, we performed static in vitro glucose-stimulated insulin\nsecretion assays on isolated islets from 3-week-old control and β-Tfrc-KO mice (manuscript\nFigure 5a-d).\nWe acknowledge that HOMA-β is an indirect measure of β-cell function and may not fully\ncapture in vivo glucose responsiveness. Therefore, we now also include the insulinogenic\nindex estimated from glucose and C-peptide measurements collected at baseline and after\n30 min. during the intraperitoneal glucose tolerance test in 6-week-old control and β-Tfrc-\nKO mice. Fasting C-peptide was slightly higher in β-Tfrc-KO mice (not statistically\nsignificant), indicative of β-cell stress. ΔC-peptide/Δglycemia (to account for glycemic\ndiserences, as per PMID: 36995380) showed that β-Tfrc-KO mice had impaired β-cell\nfunction compared to controls (Rebuttal Figure 19).\nWe included these data in the revised manuscript (revised Figure 2m).\nFigure 19: Left panel: Basal and stimulated C-peptide values during IPGTT at 6 weeks of age. Analyzed by two-way\nANOVA and uncorrected Fisher’s LSD comparison test. Right panel: Delta C-peptide–to–glucose ratio at 30 min. after\nglucose injection relative to baseline (0 min.) during IPGTT at 6 weeks of age. Minimum n=6 biologically independent\nreplicates per group and analyzed by unpaired t-test.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 22\n4. Figure 3: While Ki67 data at 3 weeks show no proliferation change, β-cell loss in Ins1-\nCre-mediated TFRC knockout (initiated embryonically) could arise from impaired\nproliferation during embryonic or postnatal stages. Quantitative assessment of β-cell\nmass and additional proliferation data at earlier developmental stages are required to\nclarify this.\nWe assessed β-cell mass and islet cell composition at postnatal day 1 and found no\ndiserences between β-Tfrc-KO and control littermates, consistent with our data at 3 weeks\nof age. We also included Ki67 immunostainings at postnatal day 1, showing no diserences\nin β-cell cycling between groups (Rebuttal Figure 20). Of note, variability in Ki67 rates at P1\nis substantial but not genotype specific. Collectively, these results indicate that embryonic\nand early postnatal β-cell proliferation is not impaired in β-Tfrc-KO mice.\nThese data are now included in the revised extended Figure 3i.\nFigure 20: Quantification of β-cell proliferation (% Ki67+NKX6.1+/NKX6.1+) at P1 and 3 weeks of age. Minimum\nn=4 biologically independent replicates per group and analyzed by multiple unpaired t-test.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 23\n5. Figure 4c: The single-cell analysis focuses exclusively on β-cells. To demonstrate β-\ncell-specific eYects and evaluate overall islet compositional changes, UMAP plots\ndepicting all islet cell types from both control and β-Tfrc-KO groups should be included.\nThank you for the suggestion. We have updated the figure accordingly (Rebuttal Figure 21\nand revised manuscript Figure 4c).\nFigure 21: Left panel, original plot: UMAP showing overlap of the β-\ncell clusters between Control (CTRL) (n=2, with 3344 β-cells) and β-\nTfrc-KO (n=2, with 2761 β-cells). Right panel, revised plot: UMAP\nshowing overlap of the islet cell clusters between CTRL (n=2, with 4682\nislet cells) and β-Tfrc-KO (n=2, with 4358 islet cells).\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 24\n6. Incomplete mechanistic validation: Although scRNA-seq and immunofluorescence\nindicate p53/CDKN1A pathway activation upon TFRC loss, functional evidence is\nlacking. Rescue experiments (e.g., p53 or HIF-1α pathway inhibition) are needed to\ndetermine whether these pathways are necessary for apoptosis or merely correlative.\nWe agree that our data demonstrate activation of the p53/Cdkn1a and HIF-1α pathways\nfollowing TFRC loss, but do not establish direct causality. While rescue experiments could\nindeed provide functional validation, in vivo inhibition of p53 is technically challenging due\nto its central role in multiple cellular processes and is considered \"undruggable\" by experts\nin this field (PMID: 36859359; PMID: 38552605). Similarly, available pharmacological HIF-1α\ninhibitors lack cell specificity in vivo, leading to substantial os-target esects (PMID:\n15930314, PMID: 35353540). For these reasons, such experiments fall beyond the scope of\nthe current study. We have therefore updated the discussion to appropriately temper our\nmechanistic conclusions.\nNonetheless, our data consistently support a mechanistic cascade in which TFRC loss\ntriggers p53-mediated β-cell apoptosis (Rebuttal Figure 22):\na) Increased DNA damage\n• Immunostaining for γH2AX (manuscript Figure 3l & extended Figure 3j):\nimmunostaining and quantitative analysis demonstrate a significant increase in\nγH2AX foci in β-cells of β-Tfrc-KO mice, indicating increased DNA double-strand\nbreaks.\nb) Activation of p53\n• Immunostaining for p53 (new data, Rebuttal figure 23): Immunostaining of\npancreas sections from 7-week-old mice shows markedly higher nuclear p53\nsignal in β-Tfrc-KO islets compared to controls.\n• scRNA-seq protein network analysis (manuscript Figure 4f): Protein-protein\ninteraction analysis (MCODE) identifies enrichment of p53 signaling pathways in β-\nTfrc-KO β-cells.\nc) Induction of p21 and cell cycle arrest\n• Immunostaining for p21 (manuscript extended Figure 4f-g): Immunostaining and\nquantification reveal increased p21 expression in β-Tfrc-KO β-cells.\n• scRNA-seq DEG analysis (manuscript Figure 4d): The Cdkn1a (encoding p21) is\nsignificantly upregulated in β-Tfrc-KO β-cells.\nd) Upregulation of pro-apoptotic genes\n• scRNA-seq analysis (new data, Rebuttal figure 24): β-Tfrc-KO β-cells exhibit higher\nexpression of canonical pro-apoptotic genes, including Bax, Puma (Bbc3), Noxa\n(Pmaip1), Perp, indicative of activation of the intrinsic apoptotic pathway.\ne) Mitochondrial dysfunction\n• Transmission electron microscopy (manuscript Figure 5e): electron micrographs\nreveal mitochondrial abnormalities, including reduced matrix density and\nelongated morphology, which are features indicative of impaired mitochondrial\nintegrity and function.\n• Seahorse mitochondrial stress test (manuscript Figure 5g-j): β-Tfrc-KO islets\nexhibited altered respiratory profiles, with significantly reduced ATP-linked\nrespiration, blunted acute glucose response, and diminished maximal respiration\ncapacity. These indicate impaired mitochondrial bioenergetics in β-Tfrc-KO islets.\n• Immunostaining for TOMM20 (novel data, Rebuttal figure 25): a marker of the\nmitochondrial outer membrane, shows decreased and fragmented mitochondrial\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 25\nstaining in β-Tfrc-KO islets, reflecting altered mitochondrial abundance and\nintegrity.\nf) Cytochrome C release\n• Immunostaining for cytochrome C (new data, Rebuttal figure 26): Confocal imaging\nreveals condensed cytosolic cytochrome C localization in β-Tfrc-KO β-cells,\nconsistent with the initiation of apoptosis.\ng) Caspase activation\n• Immunodetection of cleaved caspase 3 and caspase 9: Cleaved caspase-3 was\ntested using both immunoblotting and immunohistochemistry and was\nundetectable with antibody #9661 (Cell Signaling), even in positive control tissues.\nCaspase-9 immunostaining using ab202068 (Abcam) produced a non-specific\nsignal.\nh) Cell death\n• Immunostaining for TUNEL (manuscript Figure 3k & extended Figure 3i): TUNEL\nstaining revealed a significant increase in TUNEL+INS+ cells in β-Tfrc-KO mice,\ndemonstrating elevated β-cell apoptosis.\nFigure 22: Schematic overview of the p53-regulated cell death pathway.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 26\nFigure 23: Immunostainings for p53 (DAB, brown) and INS (immunofluorescent, green) on 7-week-old control (CTRL)\nand β-Tfrc-KO pancreas sections. n=3 biological replicates (#) per group, and per biological replicate, 4 representative\nislets are shown. Scale bar = 100 µm.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 27\nFigure 24: Average expression and percentage of β-cells expressing\nTrp53, Cdkn1a and key p53-dependent pro-apoptotic enectors (Bax,\nBbc3, Pmaip1, Perp) in β-cells from 3-week-old control (CTRL) and β-\nTfrc-KO mice.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 28\nFigure 25: Immunostaining for TOMM20 and NKX6.1 on 7-week-old control (CTRL) and β-Tfrc-KO pancreas\nsections. Scale bar = 10 µm. Yellow squares indicate the location of magnified insets, which are shown in the\nmiddle and right panel.\nFigure 26: Immunostaining for Cytochrome C and NKX6.1 on 7-week-old control (CTRL) and β-Tfrc-KO pancreas\nsections. Scale bar = 10 µm. Yellow squares indicate the location of magnified insets, which are shown in the third\nand fourth panel.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 29\n7. Figure 4: Transcriptional changes alone are insuYicient to confirm metabolic\nreprogramming. Functional validation (e.g., Seahorse glycolytic flux assays) is required\nto support claims of increased glycolytic activity\nWe agree with this point, which overlaps with Reviewer 1’s comment 3.\nHowever, measuring glycolytic activity in pancreatic islets using the Seahorse glycolytic flux\nassay is not straightforward. See below the comment from the application specialist at\nAgilent we contacted for such experiment:\n“It is diJicult – if not near impossible to measure glycolytic activity in islets using the\nSeahorse. That is because what we measure as glycolytic activity is media acidification\n(since protons generated in glycolysis are expelled from cells to the media together with\nlactate). But as you know islets are mostly comprised of beta-cells (at least in rodent islets\n– human islets are a bit more mixed cell types). Beta-cells do not really produce and\nsecrete lactate since glucose metabolism is tightly linked to mitochondrial ATP production\n– that is how they sense blood glucose. That also means that the majority of acidification\nobserved from islets is actually CO2 from mitochondrial metabolism, and that is also why\nyou will often see that the ECAR profile of a mito stress test will behave very similar to the\nOCR responses, where oligomycin will decrease ECAR (opposite most cells which will\nincrease ECAR upon oligomycin injection). Could this be the reason for why you were\nasked to look at glycolytic activity? If so, there is a perfectly good explanation for that ECAR\nresponse.\nThe best way to determine glycolytic activity is with other types of assays like Tritium\nlabeled glucose or the like.”\nTherefore, we conducted 13C-glucose-based metabolomics on isolated islets from 3-week-\nold β-Tfrc-KO and control mice. The labeling patterns showed an enrichment of glycolytic\nintermediates, with the highest fractional contribution of ¹³C atoms in D-glyceraldehyde 3-\nphosphate, pyruvic acid, and lactic acid β-Tfrc-KO islets. In addition, the m+3 isotopic\ncontribution of ¹³C in these metabolites was significantly elevated in β-Tfrc-KO islets\n(Rebuttal Figure 27). These data are direct evidence for a metabolic shift toward glycolysis\nin β-Tfrc-KO β-cells.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 30\nFigure 27: a, Overview of 13C\n6\n-glucose labeling workflow and key metabolites analyzed. b-d, Pie charts of\nthe relative abundance (RA) and fractional contribution (FC, i.e. the total percentage 13C-labeling from 13C-\nglucose) of D-glyceraldehyde, pyruvic acid, and L-lactic acid in islets isolated from control and β-Tfrc-KO\nmice. Pie radii show the RA with concentric circles corresponding from center outwards to 0.25, 0.5, 0.75\nand 1 times the largest abundance. The labeled surface fraction and percentage in the center of each pie\nreflect the FC. Analyzed by t-test for pRA and Kruskal-Wallis test for pFC using TraVis Pies. e-g, Corrected 13C\nisotopologues of D-glyceraldehyde, pyruvic acid, and L-lactic acid. Analyzed by two-way ANOVA and Šídák's\nmultiple comparisons test. n=4 biologically independent mouse islet preparations per group. *: p < 0.05, **:\np < 0.01, ****: p < 0.0001.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 31\n8. Figures 5e–f: DiYerences in insulin content may confound OCR measurements.\nSeahorse data must be normalized to total cellular protein content to account for\npotential variations in cell number/mass.\nWe agree that appropriate normalization is essential for accurate interpretation of Seahorse\ndata. While insulin content itself is unlikely to directly asect OCR measurements,\ndiserences in β-cell number or mass could asect the results. However, our previous\nanalyses demonstrated that neither islet composition nor β-cell mass is altered in β-Tfrc-KO\nmice at 3 weeks of age - the time point used for the Seahorse assay.\nTo address the reviewer’s concern, we additionally normalized the OCR data to total cellular\nprotein content using a BCA assay. This normalization did not change the overall respiration\npatterns (Rebuttal Figure 28a-b). Nonetheless, because islet insulin content is already\nreduced at this stage in our model, protein-based normalization may not accurately reflect\nfunctional cell mass, as insulin makes up much of total β-cell protein. As a further quality-\ncontrol step, we photographed each Seahorse well before and after the assay to identify\ntechnical inconsistencies (Rebuttal Figure 28c); wells in which islets had shifted away from\nthe oxygen sensor were excluded from analysis.\nTaken together, we believe that presenting respiration data normalized to the number of\nislets per well (particularly since islet size was unasected) rather than to protein content,\nprovides the most reliable and biologically meaningful comparison in this context.\nFigure 28: a, Oxygen consumption rate (OCR) (pmol/min) of 3-week-old control (CTRL) and β-Tfrc-KO islets in response\nto glucose (20 mmol/L), oligomycin (4 μmol/L), FCCP (2 μmol/L), and rotenone/antimycin A (2.5 μmol/L). b, Oxygen\nconsumption rate (OCR) (pmol/min/µg protein) of 3-week-old CTRL and β-Tfrc-KO islets in response to glucose (20\nmmol/L), oligomycin (4 μmol/L), FCCP (2 μmol/L), and rotenone/antimycin A (2.5 μmol/L). c, Imaging of wells before\nand after Seahorse assay; only wells in which islets remained stably attached to the plate surface and centered\nunderneath the oxygen sensor were included in the analysis.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 32\n9. Figure 6: The dose and frequency of iron dextran supplementation (5 mg/injection) lack\nexperimental or literature-based justification. Provide references for this regimen and\ninclude plasma iron levels post-injection to verify eYicacy.\nOur iron dextran supplementation regimen (5 mg per injection at weeks 1, 3, 5, and 7) was\nbased on published studies using comparable dosing in murine models (Rebuttal Table 1).\nThese references have now been added to the Materials and Methods section.\nThe injection schedule was designed to obtain excessive circulating non-transferrin bound\niron during critical postnatal developmental stages and into early adulthood.\nSupplementation was deliberately discontinued at 7 weeks, coinciding with the onset of\nadulthood, to assess whether β-Tfrc-KO mice could sustain physiological function and\nviability without ongoing iron supplementation, thereby providing insight into their iron\nneeds beyond developmental stages.\nTable Redacted\nTable 1: Published iron dextran supplementation regimens.\nRevised Materials and Methods section\nFor the iron supplementation experiments, control and β-Tfrc-KO mice were\nintraperitoneally injected at 1, 3, 5, and 7 weeks of age with either 25 µL phosphate-busered\nsaline (PBS) or Ferraject 200 (200 mg/mL), corresponding to a 5 mg iron dextran dose per\ninjection as in42.\nTo assess the esicacy of iron supplementation, Perls’ staining - a histochemical method for\ndetecting iron deposits - was performed on liver and pancreas sections from 12-week-old\ncontrol and β-Tfrc-KO mice that received PBS or iron (Rebuttal figure 29). Micrographs show\nsubstantial iron accumulation in the liver and in the pancreatic tissue surrounding the islets\nof mice that received iron (likely immune cells), five weeks after the final injection.\nFigure 29: Perls’ staining on liver and pancreas sections of control and β -Tfrc-KO mice injected with\nPBS or iron. Scale bar liver sections 2 mm, scale bar pancreas 100 µm and inset 2 mm.\nRevised result section:\nIron overload was confirmed by Perls’ staining on liver sections (Fig. 6d).\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 33\nTo further evaluate systemic iron status, we conducted an additional experiment in which\nC57BL6JR/j wild-type mice were injected with PBS or iron at 1, 3, 5 and 7 weeks of age. Blood\nwas collected in heparin-coated tubes at the age 8 weeks (one week after the last injection)\nfor analysis of Total Iron-Binding Capacity (TIBC), serum iron, and transferrin saturation\nusing the TIBC/Serum Iron Assay Kit (Abcam, ab239715). Repeated iron injections did not\nresult in significant changes in any circulating iron parameters at these time points (Rebuttal\nfigure 30). This likely reflects tight systemic iron regulation: excess iron is rapidly\nsequestered into storage compartments (e.g., liver, spleen, ferritin) through hepcidin-\nmediated pathways, thereby maintaining stable circulating iron levels (PMID: 35335148).\nUnfortunately, the available serum volume following cardiac puncture was insusicient to\nadditionally measure serum ferritin. We prioritized analysis of serum iron (+TIBC) as\nrequested by the reviewer. Nonetheless, Perls’ staining unequivocally demonstrated tissue\niron accumulation (Rebuttal Figure 29), confirming the esectiveness of the iron\nsupplementation regimen despite unchanged serum measurements.\nFigure 30: Serum iron, total iron-binding capacity (TIBC), and transferrin saturation measured in serum of 8-\nweek-old PBS- and iron-injected mice.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 34\n10. The rescue of β-cell defects via iron supplementation in KO mice is compelling.\nHowever, clinical context is missing: Does human data link early-life iron deficiency to\ndiabetes risk? Provide association data or discuss existing epidemiological evidence to\naddress translational implications.\nYes, human evidence supports a link between early-life iron deficiency and diabetes risk.\nEpidemiological studies show that maternal iron deficiency anemia is associated with\nimpaired fetal growth and an increased incidence of infants born small for gestational age\n(SGA) (PMID: 25946517). SGA is a well-established risk factor for type 2 diabetes later in life\n(PMID: 16615299), suggesting a potential pathway from early iron deficiency to β-cell\ndysfunction.\nGenetic evidence further supports this connection. Early case reports identified anti-TFRC\nautoantibodies in patients with diabetes (PMID: 6330553), and TFRC polymorphisms have\nbeen linked to type 2 diabetes susceptibility (PMID: 20497464). Investigation of gene-level\nassociations via HuGE Amp (https://hugeamp.org/gene.html?gene=TFRC) shows that TFRC\nvariants asect red blood cells and type 2 diabetes risk. The observation that not only HbA1c\n– which will be influenced by red blood cell turnover - but also random glucose levels are\ninfluenced by TFRC polymorphisms suggests that impaired iron handling may directly\nimpact β-cell function and glucose metabolism (Rebuttal Figure 31).\nFigure 31: Top: HuGE score–based gene-level associations showing the genetic involvement of TFRC in\nhuman diseases and traits. Bottom: zoom-in of top, showing that genetic variants in TFRC directly anect\nglucometabolic control and diabetes risk.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 35\nTogether, these findings are consistent with our observations in β-Tfrc-KO mice and\nunderscore the translational relevance of developmental iron deficiency in influencing long-\nterm metabolic disease risk.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 36\n11. Figure 7k: Tamoxifen alone reduces neonatal insulin volume, potentially confounding\nTRC knockout phenotypes. Provide structural metrics (e.g., islet number, average size)\nin neonatal tamoxifen-treated controls to isolate tamoxifen-independent eYects.\nThank you for the suggestion. We have now included the number of islets per section and\nthe average islet size in extended Figure 7g-h (also Rebuttal Figure 32). In addition, we now\nalso refer to our previous work (PMID: 26015547, from our lab) in which we showed that\ntamoxifen-mediated inhibition of ERα in the embryonic pancreas suppresses β-cell\nprogenitor expansion and proliferation, resulting in a reduced Ngn3⁺ cell number,\ndiminished β-cell replication, smaller pancreatic volume, and lower β-cell mass. These\nfindings provide a mechanistic explanation for the observed tamoxifen esect and help\ndistinguish it from specific Tfrc knock-out phenotypes.\nFigure 32: g, Quantification of average islet number per tissue section. h, Quantification of\naverage islet size (µm2). g-h, Analysis at 28 weeks of age. Minimum n=6 biologically-\nindependent replicates per group and analyzed by two-way ANOVA and Tukey's multiple\ncomparisons test.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 37\n12. While neonatal KO shows reduced β-cell iron, adult Tfrc knockout lacks direct evidence\nof intracellular iron depletion. Measure ferritin expression and labile iron pool (LIP) levels\nin adult β-cells post-knockout to confirm iron deficiency.\nWe would like to clarify that we do not claim complete intracellular iron depletion in adult β-\nTfrc-iKO mice. Our data demonstrate esicient TFRC protein deletion following tamoxifen\ninduction, confirming that the primary iron uptake pathway is disrupted. We acknowledge\nthat adult β-cells may engage compensatory mechanisms such as alternative uptake\nroutes, reduced iron demand, or iron recycling to mitigate acute iron loss.\nTo address the reviewer’s suggestion, we analyzed ferritin (FTH1/FTL1, the heavy and light\nchain respectively) expression as a proxy for intracellular iron status. Immunoblotting\nrevealed reduced ferritin protein levels at 28 weeks in adult β-Tfrc-iKO islets (16-weeks post-\ntamoxifen induction) (Rebuttal Figure 33), and immunostaining for ferritin similarly showed\ndiminished expression in adult β-Tfrc-iKO islets (Rebuttal Figure 34). The consistent\nreduction in ferritin supports impaired iron handling in β-cells lacking TFRC.\nFigure 33: Left panel: Immunoblot for FTH1/FTL1 (19-21 kDa) on 28-week-old control (CTRL) and adult β-Tfrc-\niKO islets (16-weeks TAM washout). n=3 biologically independent replicates per group. β-actin (43 kDa) served\nas loading control. Right panel: Normalized FTH1/FTL1 target signal of CTRL and β-Tfrc-iKO. Minimum n=3\nbiologically independent replicates per group and analyzed by unpaired t-test.\nFigure 34: Immunostaining for FTH1/FTL1 and\ninsulin (INS) on pancreas sections of 28-week-old\ncontrol (CTRL)+TAM and β-Tfrc-iKO+TAM (after 16\nweeks of TAM washout). Scale bar = 100 µm.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 38\nFinally, inductively coupled plasma mass spectrometry (ICP-MS) analysis of isolated islets\nfrom 28-week-old wild-type and 28-week-old (after 16w TAM wash-out) β-Tfrc-iKO+TAM\nmice provided a direct measurement of total islet iron content, confirming reduced iron\nlevels in adult β-Tfrc-iKO islets (Rebuttal figure 35).\n300\n200\n100\nR K\nC T c-i\nat c h\ne d\nW\nT\nA d\nult\nß-\nTfr\nm\ne-\ng\nA\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 39\n)telsi/gp(\neF\nFigure 35: Iron content measured by ICP-\nMS on 28-week-old wild-type and adult β-\nTfrc-iKO islets.\n13. The absence of β-cell defects in adult KO under basal conditions does not preclude\nvulnerability to metabolic stress. Assess β-cell survival/function in Tfrc-deficient\nadults exposed to high-fat diet or aging to evaluate iron’s role in stress adaptation.\nWe thank the reviewer for this valuable suggestion. We agree that assessing β-cell survival\nand function in adult β-Tfrc-iKO mice under metabolic stress - such as high-fat diet (HFD)\nexposure or aging - would provide important insights into the role of iron in β-cell stress\nadaptation. This represents a key future direction that we plan to pursue, pending funding\nand the necessary long-term experimental setup. Such studies would require large cohorts\nof age-matched transgenic animals, extended HFD exposure (≥16 weeks), and longitudinal\nmetabolic follow-up, which are beyond the scope and timeline of the current revision.\nNonetheless, our current findings - demonstrating preserved β-cell mass and function in\nadult β-Tfrc-iKO mice under basal conditions - support the conclusion that TFRC is essential\nfor β-cell survival during development but dispensable in adult β-cells in the absence of\nmetabolic stress.\nWe have incorporated this perspective into the revised discussion, emphasizing the\nimportance of future studies examining iron homeostasis under metabolic stress conditions\nsuch as HFD exposure and aging. These experiments will form the basis of a broader\nresearch project aimed at dissecting the interplay between iron metabolism and β-cell\nvulnerability. In addition to studies in adult β-Tfrc-iKO mice under HFD and aging, we plan to\ninvestigate metabolic stress in β-Tfrc-heterozygous-KO (β-Tfrc-hz-KO) mice, which remain\nnormoglycemic for up to one year under basal conditions (Rebuttal Figure 36). This project\nwill also include dietary iron modulation to better understand the contribution of systemic\niron availability to β-cell adaptation and glucose homeostasis. Such investigations may have\ntranslational relevance, given established links between iron status and type 2 diabetes in\nhumans, as supported by GWAS data and HuGE plots (Rebuttal reviewer 2 comment 9).\n200\n100\n0\n4 8 1216202428323640444852\nAge (weeks)\nFigure 36: Longitudinal monitoring of non-fasting blood glucose (mg/dL) in β-Tfrc\nheterozygous (hz)-KO mice over 52 weeks. Data represent a minimum of n=12\nbiologically-independent replicates.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 40\n)Ld/gm(\naimecylg\ngnitsaf-noN\nβ-Tfrcfl/WT (hz) -KO\nReviewer #3\nThis study investigates the role of iron deficiency in maturation and function of pancreatic β cells.\nThe authors observed that expression of murine transferrin receptor 1 (Tfrc) is relatively high\npostnatally but slowly declines with age. Mice with targeted pancreatic β cell-specific disruption\nof Tfrc developed hyperglycemia and diabetes due to progressive apoptotic death of β cells,\nfollowing mitochondrial dysfunction and switch to glycolytic energy metabolism. This phenotype\nwas only observed when Tfrc was eliminated during early postnatal development but not in adult\nmice and could be rescued by parenteral supplementation of excess iron. The authors conclude\nthat iron starvation is detrimental to pancreatic β cells during development but not in the adult\nstage and provide supportive evidence using a human iPSC β cell aggregate model.\nThe findings are novel and oser significant insights on the role of iron in pancreatic β cell function\nand pathophysiology. The data are of high technical quality and their interpretation is appropriate.\nThe manuscript would further improve by addressing following issues:\nComments of the reviewer and answers of authors:\n1. It will be important to assess the iron status of pancreatic β cells in various\nexperimental settings: In constitutive Tfrc knockouts before and after iron\nsupplementation, as well as in temporal Tfrc knockouts in young and adult mice.\nSome iron markers are shown, but not all of them are specific (for instance Slc40a1\nmRNA). A comprehensive assessment of iron status can be done by measuring ferritin\nby Western (as done in Figures 1g and 2b), ferroportin by Western and/or\nimmunohistochemistry, labile iron by a fluorescent probe, and (if possible) IRP2\nexpression or IRP activity.\nWe thank the reviewer for this valuable suggestion. Due to the limited islet yield from\nneonatal β-Tfrc-iKO and from β-Tfrc-KO+PBS mice (the control group in the iron rescue\nexperiments), immunoblot analyses could not be performed in these specific models.\nTo address this limitation, we evaluated ferritin expression in formalin-fixed parasin-\nembedded (FFPE) pancreatic sections across all relevant models, thereby providing a\nbroader evaluation of islet iron status (Rebuttal Figure 37). In control tissues across\nmultiple experimental settings, ferritin expression (assessed by FTH1 & FTL1) increases\nwith age. Ferritin levels are reduced in β-Tfrc-KO at 3 weeks compared age-matched\ncontrols. In the rescue experiments, β-Tfrc-KO+PBS also exhibit reduced ferritin levels,\nwhereas β-Tfrc-KO+iron mice show a slight increase relative to β-Tfrc-KO+PBS, yet their\nlevels remain lower than those of CTRL+PBS and CTRL+iron. Following inducible Tfrc-\ndeletion, ferritin expression is also decreased in the neonatal model (neonatal β-Tfrc-iKO)\nassessed at 3 weeks of age and in adult model (adult β-Tfrc-iKO) assessed at 28 weeks of\nage.\nIn addition, we performed immunoblot analysis for iron regulatory protein 2 (IRP2) on DFO-\nor FeCitr-treated islets and 3-week-old CTRL and β-Tfrc-KO to assess IRP activity (Rebuttal\nFigure 38). As expected, iron deficiency (either chemical or genetic) increases expression\nof the iron sensor IRP2, while iron supplementation decreases its expression.\nFinally, total iron content was quantified by Inductively Coupled Plasma-Mass\nSpectrometry (ICP-MS) in β-Tfrc-KO and adult β-Tfrc-iKO islets (Rebuttal Figure 39), which\nwas reduced in both setting.\nTogether, these complementary approaches oser a comprehensive evaluation of β-cell\niron status across the diserent experimental settings.\nAn overview is presented in the table below:\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 41\nModel Immunostaining Immunoblot ICP-MS\nfor ferritin\nβ-Tfrc-KO Yes Yes (FTH1/FTL1 & IRP2) Yes\nβ-Tfrc-KO Yes Not possible due to low Not possible due to low\n+PBS/iron islet yield (in β-Tfrc-KO islet yield (in β-Tfrc-KO\n+PBS) +PBS)\nNeonatal Yes Not possible due to low Not possible due to low\nβ-Tfrc-iKO islet yield islet yield\nAdult Yes Yes (FTH1/FTL1) Yes\nβ-Tfrc-iKO\nChemical iron / Yes (FTH1/FTL1 /\nmodulation & IRP2)\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 42\nFigure 37: Immunostaining for FTH1/FTL1 and insulin (INS) on pancreas sections of 3-week-old control (CTRL)\nand β-Tfrc-KO (top); 12-week-old CTRL and β-Tfrc-KO that received PBS or iron (middle); 3-week-old CTRL and\nneonatal β-Tfrc-iKO that received tamoxifen (bottom left); and 28-week-old CTRL and adult β-Tfrc-iKO that\nreceived tamoxifen (TAM) (bottom right). Scale bar = 100 µm.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 43\nFigure 38: Top left: Immunoblot for IRP2 (105 kDa) on control (CTRL), DFO- and FeCitr treated mouse islets.\nn=3 biologically independent replicates per group. The total protein stain served as loading control. Top right:\nNormalized IRP2 target signal of CTRL, DFO- and FeCitr-treated mouse islets. n=3 biologically independent\nreplicates per group and analyzed by one-way ANOVA and Dunnett's multiple comparisons test. Bottom left:\nImmunoblot for IRP2 (105 kDa) on 3-week-old CTRL and β-Tfrc-KO islets. n=3 biologically independent\nreplicates per group. The total protein stain served as loading control. Bottom right: Normalized IRP2 target\nsignal of and β-Tfrc-KO islets. n=3 biologically independent replicates per group and analyzed by unpaired t-\ntest.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 44\n100\n80\n60\n40\n20\nTfr\nß-\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 45\n)telsi/gp(\neF\n300\n200\n100\nat c h\ne d\nW\nT\nA d\nult\nß-\nTfr\nm\ne-\ng\nA\n)telsi/gp(\neF\nFigure 39: ICP-MS analysis of islet iron content. Left panel: 3-week-old CTRL & β-Tfrc-KO islets. Right\npanel: 28-week-old CTRL & adult β-Tfrc-iKO islets. n=3 biologically-independent replicates per group.\nCross symbols indicate measurements below the detection limit.\n2. Which is the recombination eYiciency for Tfrc ablation in β cells? It appears low\ncompared to that in the YFP reporter mouse model in Fig. 1. Why is there only 50%\nreduction in Tfrc protein and which are the Tfrc mRNA levels?\nThe yellow fluorescent protein (YFP) reporter expression observed in Ins1-Cre; R26RYFP\nsections (manuscript extended Figure 1g, or Rebuttal Figure 40, left) closely parallels that\nin β-Tfrc-KO sections (manuscript Figure 3g, or Rebuttal Figure 40, right), confirming\nesicient recombination in β-cells. In β-Tfrc-KO mice, the few remaining insulin-positive\ncells that remain consistently co-express YFP, further supporting β-cell-specific\nrecombination.\nFigure 40: Left panel: Representative images of immunofluorescent staining for insulin (INS)\nand yellow fluorescent protein (YFP) on sections of β-cell reporter (Ins1-Cre; R26RYFP) pancreas.\nScale bar = 100 µm. Right panel: Representative images of immunofluorescent staining for INS,\nglucacon (GCG), somatostatin (SST), and YFP on sections of 7-week-old control (CTRL) and β-\nTfrc-KO pancreas. Scale bar = 100 µm.\nAt the transcript level, Tfrc mRNA is reduced by ~50% in β-Tfrc-KO islets (fold change:\n1.044 in controls vs. 0.564 in β-Tfrc-KO islets; manuscript extended Figure 4h). Since islets\ncontain a substantial proportion of non-β endocrine cells (α-cells ~28.8% and δ-cells\n~19.2%, see quantification in manuscript Figure 3f) that retain Tfrc expression, this\naccounts for the partial reduction observed in mRNA and protein levels when analyzed in\nwhole-islet lysates. Importantly, in YFP-sorted β-cells from 3-week-old mice, Tfrc\nexpression was reduced by ~3-fold in β-Tfrc-KO compared with control β-cells (Rebuttal\nFigure 41).\n0.20\n0.15\n0.10\n0.05\n0.00\nCTRL ß-Tfrc-KO\nß-cells ß-cells\nFigure 41: Tfrc expression in sorted YFP+ β-cells of\nIns1-Cre; R26RYFP(CTRL) and β-Tfrc-KO mice. Minimum\nn=3 biological replicates and analyzed by unpaired t-test.\nThe ~50% reduction in TFRC protein levels observed in β-Tfrc-KO islets (manuscript figure\n2b) reflects both cellular heterogeneity and the post-translational regulation of TFRC by\niron availability. Under iron-replete conditions, TFRC is rerouted from the canonical\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 46\n)tC\n-2(\nnoisserpxe\nevitaleR\nTfrc\nclathrin/AP2-mediated recycling pathway to lysosomal degradation, thereby lowering\nsurface TFRC levels and limiting further iron uptake (PMID: 33236955). This iron-induced\nswitch serves as a homeostatic mechanism to prevent iron overload. In contrast, under\niron-deficient (or basal) conditions, TFRC is preferentially recycled back to the plasma\nmembrane to sustain transferrin-bound iron uptake. This regulatory mechanism may also\ncontribute to the partial reduction in TFRC protein levels observed in β-Tfrc-KO islets at 3\nweeks of age, depending on the local iron status within the islet microenvironment.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 47\n3. In line 200, it is stated that Gapdh “facilitates iron uptake through noncanonical\nsiderophore interactions (ref 23)”. My understanding is that according to work by the\nauthors of the review article in ref. 23, Gapdh may function as a moonlighting\ntransferrin receptor, with much lower aYinity for transferrin than Tfrc.\nThank you for this comment. We have revised the text in the manuscript accordingly.\nRevised:\n“Other upregulated genes included glyceraldehyde-3-phosphate dehydrogenase\n(Gapdh), which can act as a moonlighting transferrin receptor23, and Calcium Voltage-\nGated Channel Subunit Alpha1 C (Cacna1c), an L-type calcium channel involved in non-\ntransferrin-bound iron uptake7,24, suggesting compensatory mechanisms to circumvent\nTFRC loss (Fig. 4d).”\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 48\n4. In the KEGG pathway analysis (Fig. 4e), the first hit is “coronavirus disease”. What\ndoes this mean?\nThe enrichment of the “Coronavirus disease (COVID-19)” pathway (KEGG:05171) in\nmanuscript Figure 4e does not indicate viral infection but instead reflects upregulation of\ngenes associated with innate immune, inflammatory, and cellular stress responses. Such\npathways are broadly activated under diverse stress conditions, including metabolic and\ntranslational stress.\nNotably, KEGG:05171 includes many cytoplasmic ribosomal protein genes (e.g., RPL37,\nMAPK1, RPL41, TAB2, RPLP2, RPS15A), which also appear in “Ribosome” pathway\n(KEGG:03010), the second top hit in our KEGG pathway analysis. There is substantial\noverlap between the two pathways, with 61 of 68 genes shared; the remaining few\n(MRPL17, MRPS21, MRPL28) correspond to mitochondrial ribosomal proteins.\nThese findings indicate that our dataset is enriched for both cytoplasmic and\nmitochondrial ribosomal subunits, consistent with translational and mitochondrial stress\nor compensatory adaptation in Tfrc-deficient β-cells, where iron-dependent mitochondrial\nfunction is impaired. Iron deficiency is known to disrupt mitochondrial translation and can\ntrigger upregulation of both cytoplasmic and mitochondrial ribosomal components as part\nof a broader stress response.\nWe have now included a sentence in the Results section to explain why this KEGG pathway\nis upregulated.\nRevised:\n“KEGG pathway analysis of upregulated genes revealed enrichment in HIF-1 signaling,\ninsulin secretion, and glycolysis, indicating a metabolic shift (Fig. 4e). Notably, the\nenrichment of the “Coronavirus disease” KEGG pathway reflects increased expression of\ncytoplasmic and mitochondrial ribosomal proteins – canonical components of cellular\nstress responses.”\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 49\n5. The data in Fig. 5e-h imply a shift from oxidative phosphorylation to glycolysis.\nDirect assessment of extracellular acidification rates (ECAR) would strengthen this\nconclusion.\nBased on our mitochondrial stress test assay, β-Tfrc-KO islets show a marked reduction in\noxygen consumption rate (OCR) compared with controls, consistent with impaired\nmitochondrial respiration (manuscript Figure 5g). Interestingly, this decline in OCR is not\nmatched by a proportional reduction in extracellular acidification rates (ECAR) within the\nsame assay, suggesting that glycolytic flux contributes relatively more to the metabolic\nprofile of β-Tfrc-KO islets (Rebuttal Figure 42).\nFigure 42: Extracellular acidification rate (ECAR) (mpH/min) of 3-\nweek-old control (CTRL) and β-Tfrc-KO islets in response to\nglucose (20 mmol/L), oligomycin (4 μmol/L), FCCP (2 μmol/L),\nand rotenone/antimycin A (2.5 μmol/L).\nWhile our data suggested a relative shift toward glycolytic metabolism, we agree that we\ndid not provide definitive evidence in our original submission. Measuring glycolytic activity\nin pancreatic islets with glycolytic flux Seahorse assays, however, is not recommended\n(see also Reviewer 2, comment 7).\nTo provide complementary evidence, we conducted 13C-glucose-based metabolomics on\nisolated islets from 3-week-old β-Tfrc-KO and control mice. The labeling patterns showed\nan enrichment of glycolytic intermediates, with the highest fractional contribution of ¹³C\natoms in D-glyceraldehyde 3-phosphate, pyruvic acid, and lactic acid β-Tfrc-KO islets. In\naddition, the m+3 isotopic\ncontribution of ¹³C in these metabolites was significantly elevated in β-Tfrc-KO islets\n(Rebuttal Figure 43). These data are direct evidence for a metabolic shift toward glycolysis\nin β-Tfrc-KO β-cells.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 50\nFigure 43: a, Overview of 13C-glucose labeling workflow and key metabolites analyzed. b-d, Pie charts of the relative\n6\nabundance (RA) and fractional contribution (FC, i.e. the total percentage 13C-labeling from 13C-glucose) of D-\nglyceraldehyde, pyruvic acid, and L-lactic acid in islets isolated from control and β-Tfrc-KO mice. Pie radii show the\nRA with concentric circles corresponding from center outwards to 0.25, 0.5, 0.75 and 1 times the largest abundance.\nThe labeled surface fraction and percentage in the center of each pie reflect the FC. Analyzed by t-test for pRA and\nKruskal-Wallis test for pFC using TraVis Pies. e-g, Corrected 13C isotopologues of D-glyceraldehyde, pyruvic acid, and\nL-lactic acid. Analyzed by two-way ANOVA and Šídák's multiple comparisons test. n=4 biologically independent\nmouse islet preparations per group. *: p < 0.05, **: p < 0.01, ****: p < 0.0001.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 51\n6. Is the decrease in mature insulin granules of knockout mice shown in Fig. 5j\nstatistically significant?\nThis question aligns with Reviewer 1, comment 9.\nIn the original comparison, the difference was not statistically significant: an unpaired t-\ntest comparing control (n = 5, mean = 0.0016) and β-Tfrc-KO (n = 8, mean = 0.0013) groups\nyielded a p-value of 0.1077. However, this reflected variability among β-Tfrc-KO cells at 4\nweeks, with some showing clear signs of iron deficiency-induced damage (affected cells),\nwhile others were still intact (unaffected cells). To better capture the heterogeneity at this\ndisease stage, we subdivided the β-Tfrc-KO group in affected and unaffected β-cells\n(based on the criterium of cell membrane detachment) revealing that affected cells\ncontained significantly fewer mature granules than both unaffected and control β-cells\n(Rebuttal Figure 44).\nFigure 44: Left panel: Representative electron micrographs of 4-week-old control (CTRL) and β-Tfrc-KO islets,\nwith β-Tfrc-KO islets further divided into unanected and anected β-cells. Yellow arrows indicate cell membrane\ndetachment and mitochondrial abnormalities in β-Tfrc-KO islets. Upper panel: scale bar = 5 µm. Lower panel:\nscale bar = 1 µm. Right panel: Quantification of the number of mature insulin granules per cytosol area,\nanalyzed by one-way ANOVA and Tukey's multiple comparisons test.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 52\n7. Can the phenotype of β-cell-specific Tfrc knockout mice be rescued by iron dextran\ninjections starting at later time points (in adult animals)?\nWe thank the reviewer for this experimental suggestion. To assess whether β-cell\ndysfunction in β-Tfrc-KO mice can be reversed by iron supplementation at later stages, we\nadministered a single 5 mg iron dextran injection to control and β-Tfrc-KO mice at 7 weeks\nof age and monitored body weight and non-fasting glycemia weekly until 12 weeks. Only\ndiabetic β-Tfrc-KO mice with random glycemia > 500 mg/dL at 6 weeks were included.\nOf note, mice showed signs of distress from the onset of the experiment which aggravated\nduring follow-up. A single iron injection at 7 weeks of age produced a modest improvement\nin glycemia: mean non-fasting glucose decreased from 576.5 mg/dL at 7 weeks to 466.5\nmg/dL at 12 weeks in β-Tfrc-KO. One of four β-Tfrc-KO mice showed a decrease in glycemia\nafter iron administration, but the majority remained severely hyperglycemic (> 400 mg/dL)\nand for animal welfare reasons we terminated the experiment (Rebuttal Figure 45).\nThese findings indicate that iron supplementation initiated after diabetes onset has limited\nesicacy in rescuing the β-Tfrc-KO phenotype, suggesting that β-cell defects caused by Tfrc\nloss are largely irreversible once established.\nFigure 45: Non-fasting blood glucose levels\n(mg/dL) of control (CTRL) and β-Tfrc-KO mice\nfrom 6 to 12 weeks of age. One single iron\ninjection was given at 7 weeks of age.\nMinimum n=4 biologically independent\nreplicates per group.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 53\n8. In Fig. 6b-c, what does the discrepancy in Slc39a14 expression between the\nscRNASeq and qPCR data mean?\nThe apparent discrepancy between scRNA-seq and qPCR results likely reflects the\ncellular resolution of these approaches. The scRNA-seq analysis captures gene\nexpression specifically in β-cells, where we observed an upregulation of Slc39a14. In\ncontrast, the initial qPCR was performed on whole islets, which include other endocrine\n(α-, δ-cells) and non-endocrine cell types, potentially masking β-cell-specific changes.\nTo directly assess Slc39a14 expression in β-cells, we performed qPCR on YFP+ sorted β-\ncell fractions from β-cell reporter (control) and β-Tfrc-KO mice. In these purified β-cells,\nSlc39a14 expression was ~2-fold higher in β-Tfrc-KO β-cells, confirming that Tfrc-\nknockout in β-cells induces upregulation of other iron importers.\nWe now report Slc39a14 and Slc11a2 expression by qPCR in YFP+ sorted β-cells,\nrather\nthan in whole islets in the manuscript (Rebuttal Figure 46).\nCTRL\nβ-Tfrc-KO\nc\nβ-cells (3wks)\nSlc11a2\nFigure 46: Revised Figure 6C of the manuscript. Left panel: qRT-PCR\nanalysis of Slc11a2 and Slc39a14 expression in islets of 3-week-old\ncontrol (CTRL) and β-Tfrc-KO. Expression levels are normalized to\nSdha. n=3 biologically independent mouse islet preparations per\ngroup. Right panel: qRT-PCR analysis of Slc11a2 and Slc39a14\nexpression in YFP+ β-cells of 3-week-old CTRL and β-Tfrc-KO.\nExpression levels are normalized to Sdha. Minimum n=3 biologically-\nindependent mouse islet preparations per group.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 54\n-\ntC 0.6\n0.4\n0.2\n0.0\n)\n2(\nnoisserpxe\nevitaleR\nc\nSlc39a14\n2.5 0.10\n2.0 0.08\n1.5 0.06\n1.0 0.04\n0.5 0.02\n0.0 0.0\nSlc11a2 Slc39a14\nnoissserpxe\nANRm\nevitaleR\n9. The iron-dependent induction of DMT1 in Tfrc-deficient β cells (Fig. 6n) is very\ninteresting. DMT1 has been reported to mediate excess iron uptake by these cells\n(Hansen et al, Cell Metab, 2012; PMID 23000401).\nWe thank the reviewer for this insightful comment. Hansen et al. (Cell Metab, 2012; PMID\n23000401) reported that exposure to inflammatory cytokines induces Dmt1 expression in\nβ cells, promoting excessive iron uptake, reactive oxygen species (ROS) production, and β\ncell damage.\nIn our study, we observed that Tfrc-deficient β-cells upregulate DMT1 in an iron-\ndependent manner, consistent with an adaptive response to impaired Tfrc-mediated iron\nuptake. This compensatory induction likely helps maintain intracellular iron levels through\nalternative import routes under iron-limiting or stress conditions. Notably, our data show\nthat DMT1 upregulation in this context is responsive to extracellular iron levels,\nhighlighting its regulatory flexibility and iron-sensing capacity.\nThese findings extend those of Hansen et al. by suggesting that DMT1 is not only a\nmediator of iron-induced toxicity but also a dynamically regulated component of iron\nacquisition when canonical Tfrc-mediated iron import is disrupted. We now emphasize\nthis point in the revised Results section and reference to the study by Hansen et al.\nRevised:\n“Notably, DMT1 – a transporter implicated in inflammation-induced iron toxicity27 and\ndynamically regulated during iron acquisition – was upregulated in both PBS- and iron-\ntreated β-Tfrc-KO islets, with the former retaining few β-cells and the latter maintaining\nnormal β-cell numbers (Fig. 6o).”\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 55\n10. In line 272, how much is “eYicient recombination”?\nWe defined “esicient recombination” quantitatively. By immunoblot, TFRC protein is\nreduced ~4-fold in adult β-Tfrc-KO+TAM versus control+TAM islets.\nIn addition, immunostaining of pancreatic sections and quantification of YFP+ cells among\nINS+ β-cells (n=3 biological replicates) revealed an average of 90.91 ± 4.58% YFP+(/INS+)\ncells, indicating recombination in ~91% of β-cells. Together, these data confirm esicient β-\ncell recombination. This quantification is now included in the revised manuscript (Rebuttal\nFigure 47).\nc\nINS YFP\n100\n50\nimmunofluorescent staining for INS and yellow\nfluorescent protein (YFP) on sections of adult β-\nTfrc-iKO pancreas and quantification of the\npercentage of YFP+ β-cells. Scale bar = 100 µm.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 56\n)sllec\n+SNI%(\nsllec\n+PFY\n11. Lines 282-284: Another possibility is that mature β cells have limited iron eYlux\ncapabilities due to low ferroportin expression and thus retain more iron.\nWe thank the reviewer for this comment. Indeed, our bulk RNA sequencing data\n(manuscript Figure 1c, or below Rebuttal Figure 48) from wild-type sorted islet cells across\ndiserent ages show that adult and aged β-cells exhibit reduced expression of the iron\nexporter Slc40a1 (ferroportin). We have added a sentence in the revised Results section\nto highlight this observation.\nFigure 48: Heatmap showing the dynamics of iron metabolism-related\ngene expression over the β-cell life span, and the static expression of\nthese genes in α- and δ-cells. Clusters are generated by the dendrogram\ncut, dotted horizontal line, and color code above. Slc40a1 = Ferroportin.\nRevised:\n“This suggests that with maturation β-cells either acquire compensatory iron uptake\nmechanisms, exhibit lower iron eslux, reduce iron demands, or recycle intracellular iron\nmore esiciently, ultimately reducing their dependence on TFRC-mediated iron uptake.”\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 57\n12. As noted in the Discussion, the responses of Tfrc following treatment of cells with iron\nor DFO are expected; therefore, some of these data could be shown in the\nsupplement, if space is limiting.\nWe agree that some of the observed changes in Tfrc expression are expected, reflecting\nthe established IRE/IRP system that regulates cellular iron homeostasis. However, our\ndata show that these regulatory shifts are largely restricted to β-cells (as assessed using\nβ-cell reporter mice), underscoring their high requirement for iron compared with\nneighboring endocrine cells. This cell-type specificity provides new biological insight and\njustifies – in our opinion - inclusion in the main figure. Nevertheless, we are open to\ndiscuss relocating the data to the supplement with the editor, should space constraints\narise.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 58\n13. Lines 366-368: “…iron supplementation rescued β cell loss, indicating that alternative\niron uptake pathways can compensate when transferrin-mediated delivery is\nimpaired”. This is very likely true; however, there is significant residual Tfrc\nexpression, which may contribute to the rescue when transferrin becomes fully\nsaturated (following iron dextran injections). “Enhanced Tfrc recycling” (line 403)\ncould also be a factor.\nWe agree that residual Tfrc expression, along with its stabilization or enhanced recycling\nunder iron-deficient conditions, may contribute to the observed rescue of β cell loss\nfollowing iron dextran administration. To address the reviewer’s concern, we revised the\ntext in the indicated lines by softening our conclusion and replacing “indicating” with\n“suggesting.”\nRevised:\nRemarkably, iron supplementation rescued β-cell loss, suggesting that alternative iron\nuptake pathways can compensate when transferrin-mediated delivery is impaired.\nRemarks to author on “Iron deficiency induces maturation-dependent loss of pancreatic β-cells.” 59\nPoint-by-point response to the reviewer’s comments\n“Iron deficiency induces maturation-dependent loss of pancreatic β-cells.”\nAnnelore Van Mulders1, Lien Willems1, Sophie Coenen1, Stephanie Bourgeois1, Xiaoyan Yi2,\nYue Tong2, Gunter Leuckx1,£, Yves Heremans1, Julie Pierreux1, Laure Degroote1, Toshiaki\nSawatani2, Chiara Vinci2, Gamze Ates3, Ann Massie3, Françoise Carlotti4, Eelco de\nKoning4, Thomas Mandrup-Poulsen5, Clara Zelinsky6, Steven Goderis6, Bart Ghesquière7,\nRaphael Scharfmann8, Miriam Cnop2,9,10, Nico De Leu1,11,12,*, Willem Staels1,13,*.\nReviewers' comments\n Reviewer #1 (Remarks to the Author): The authors have addressed all of my\ncomments comprehensively, and I have no further comments. I believe that the\ncurrent version of the manuscript is ready for publication.\n Reviewer #2 (Remarks to the Author): The authors have done a great job with the\ncareful revision of this manuscript. The study has been substantially improved, and all\nof my previous concerns have been fully addressed.\n Reviewer #3 (Remarks to the Author): The revised manuscript has addressed all\nissues raised by the reviewers in a satisfactory manner\nResponse of authors\n Answer to reviewers 1-3: We thank the reviewers for their positive feedback and are\npleased that they find the revised manuscript satisfactory. No further changes were\nrequested."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all of my comments comprehensively, and I have no further comments. I believe that the current version of the manuscript is ready for publication.", "reviewer_2": "The authors have done a great job with the careful revision of this manuscript. The study has been substantially improved, and all of my previous concerns have been fully addressed.", "reviewer_3": "The revised manuscript has addressed all issues raised by the reviewers in a satisfactory manner"}, "author_response": "Point-by-point response to the reviewer’s comments\n“Iron deficiency induces maturation-dependent loss of pancreatic β-cells.”\nAnnelore Van Mulders1, Lien Willems1, Sophie Coenen1, Stephanie Bourgeois1, Xiaoyan Yi2,\nYue Tong2, Gunter Leuckx1,£, Yves Heremans1, Julie Pierreux1, Laure Degroote1, Toshiaki\nSawatani2, Chiara Vinci2, Gamze Ates3, Ann Massie3, Françoise Carlotti4, Eelco de\nKoning4, Thomas Mandrup-Poulsen5, Clara Zelinsky6, Steven Goderis6, Bart Ghesquière7,\nRaphael Scharfmann8, Miriam Cnop2,9,10, Nico De Leu1,11,12,*, Willem Staels1,13,*.\nReviewers' comments\n Reviewer #1 (Remarks to the Author): The authors have addressed all of my\ncomments comprehensively, and I have no further comments. I believe that the\ncurrent version of the manuscript is ready for publication.\n Reviewer #2 (Remarks to the Author): The authors have done a great job with the\ncareful revision of this manuscript. The study has been substantially improved, and all\nof my previous concerns have been fully addressed.\n Reviewer #3 (Remarks to the Author): The revised manuscript has addressed all\nissues raised by the reviewers in a satisfactory manner\nResponse of authors\n Answer to reviewers 1-3: We thank the reviewers for their positive feedback and are\npleased that they find the revised manuscript satisfactory. No further changes were\nrequested."}]

Decision letter