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. Author manuscript; available in PMC: 2026 Jul 8.
Published in final edited form as: J Nutr Biochem. 2026 Jun 13;157:110448. doi: 10.1016/j.jnutbio.2026.110448

Moderate iron restriction improves metabolism via epigenetic regulation of GDF15

Jinying Yang 1,2,3,9, Limin Shi 1,2,3,9, Anna L Cubito 1, Akshaya Govarthanan 1, Jian S Sabripour 1, Karen Scott 3,4, Annette de Kloet 3,5, Feng Yue 6, Bin Liu 1,2, Larissa J Strath 7,8, Eric Krause 3,4, James F Collins 1,2, Zhiyong Cheng 1,2,3
PMCID: PMC13338862  NIHMSID: NIHMS2188579  PMID: 42288146

Abstract

Iron overload disrupts endocrine function and metabolic health, while iron chelation and phlebotomy enhance metabolic fitness in humans and mice. However, the working mechanism of iron-lowering strategies remains largely undefined, and it is unclear whether dietary iron restriction can serve as a new strategy to treat metabolic syndrome. Here we show that 20-ppm iron (i.e., iron moderately restricted) diet increased insulin sensitivity, adipose mitochondrial biogenesis, and energy expenditure compared to 50-ppm iron (i.e., iron adequate) diet in mice. By contrast, severe iron restriction (5-ppm iron diet) caused anemia, underweight and metabolic disorder. Mechanistically, moderate iron restriction induced a condition of subcellular “iron deficiency” due to iron redistribution into mitochondria in adipose tissues, which augmented H3K4 methylation possibly by suppressing iron-dependent histone demethylase like JARID. Enriched H3K4 methylation upregulated the expression of GDF15, a nutrient sensor that promotes adipose browning and metabolic enhancement. Pharmacological inhibition of H3K4 methylation or knockdown of GDF15 prevented iron restriction-induced enhancement of insulin sensitivity. Our study reveals a potential strategy targeting dietary iron to prevent metabolic disorder. It provides the first line of evidence of epigenetic regulation of GDF15 via an iron restriction-H3K4 methylation cascade. Future studies of the H3K4 methylation-GDF15 axis may fuel developing therapeutic options or dietary interventions for metabolic disease.

Keywords: moderate iron restriction, adipose browning, GDF15, epigenetic regulation, metabolic health

Graphical Abstract

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INTRODUCTION

Overnutrition may disrupt endocrine function and metabolic fitness, which includes both macronutrients and micronutrients [1-4]. Excess iron induces oxidative stress that causes insulin resistance or insulin deficiency [3, 4]. High dietary iron intake dysregulates adipokines leptin and adiponectin, increasing inflammation and reducing insulin sensitivity, the hallmarks of diabetes and obesity [5, 6]. Moreover, overproduction of ferrous iron activates NF-kB and induces insulin resistance in mice [7]. In children with obesity, iron overload elevates blood glucose [8], and type 1 diabetes risk increases in children exposed to maternal iron supplementation [9]. Thus, control of iron status is critical for metabolic health.

To treat metabolic disorders, iron-lowering strategies, such as iron chelation and phlebotomy, have been developed to improve glycemic control in humans and mice [10-15]. Phlebotomy improves insulin sensitivity and β-cell function and attenuates vascular dysfunction in type 2 diabetic patients [12, 13]. Even for human subjects with normal glucose tolerance, blood donation significantly reduced diabetes marker HbA1c [16]. Iron chelation protects obese mice from β-cell dysfunction and diabetes by mitigating oxidative stress and inflammation [10, 11]. However, the mechanism of iron-restriction induced metabolic enhancement is largely unknown, and it remains to be defined whether dietary iron restriction can serve as a new strategy to battle metabolic disease.

In this study we develop a strategy of dietary iron restriction and discover a novel epigenetic pathway, by which moderate restriction induces adipose browning and improves metabolism in mice via growth differentiation factor 15 (GDF15). Moderate restriction results in adipose iron redistribution into mitochondria and constitutes a condition of subcellular “iron deficiency”, which mitigates the activity of histone demethylase, jumonji domain ARID-containing protein (JARID). This enriches H3K4 methylation (H3K4me) and upregulates GDF15, a nutrient sensor that induces adipose browning and metabolic improvement [17-19]. Pharmacological inhibition of H3K4 methylation or silencing GDF15 in adipocytes abolishes the beneficial effects of iron restriction. We also show that severe iron restriction causes anemia, underweight, and metabolic disorder. Our study provides the first line of evidence that demonstrates epigenetic regulation of GDF15 via an iron restriction-H3K4 methylation cascade, opening a new window for future studies to develop epigenetic therapy and dietary intervention for metabolic disease.

RESULTS

Moderate iron restriction improves metabolism in mice.

According to the National Research Council [20], 50 ppm dietary iron indicates physiologically adequate iron supply for rodents, and 20 ppm dietary iron represents moderate iron restriction [21]. Male C57BL/6 mice fed on 20 ppm iron high fat diet (HFD) for 20-22 weeks had lower body weight and fat mass, but lean mass increased in comparison to mice C57BL/6 mice fed on 50 ppm iron HFD (Figure 1, A-C). The mice did not develop anemia (Figure 1D), although hemoglobin was significantly reduced by moderate iron restriction (13.6 vs 16.1 g/dL in mice on 50 ppm iron HFD, p<0.05), which is considered as normal (hemoglobin >=13.6 g/dL) [22]. The mice on 20 ppm iron HFD had greater food intake (Figure 1E), glucose clearance capacity (Figure 1, F-G), and insulin sensitivity (Figure 1, H-I). Respiratory quotient (RQ) revealed a shift toward 1.0, suggesting that more fat was catabolized for energy metabolism (Figure 1, J-K). Indeed, energy expenditure was significantly increased in mice on 20 ppm versus 50 ppm iron HFD (Figure 1, L-M), concurrent with higher O2 consumption (Figure 1, N-O) and CO2 production (Figure 1, P-Q). As observed in males, 20 ppm iron diet improved metabolism and adiposity in females (Figure 1s), suggesting that moderate iron restriction induces metabolic enhancement in both sexes. However, mice on 4 ppm iron HFD for only 6 weeks (Figure 2s) developed very severe anemia (6.5 g/dL hemoglobin) according to the American Association for Laboratory Animal Science (severe anemia: 7.3-9.0 g/dL hemoglobin) [22]. These data suggest that moderate, but not severe, iron restriction from diet benefits metabolic health in mice. Prolonged or infinite moderate iron restriction, however, can be harmful as it will eventually cause severe iron deficiency and anemia.

Figure 1.

Figure 1.

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Moderate iron restriction enhances energy expenditure and metabolic health. (A-E) The effects of 20-ppm vs 50 ppm iron high fat (60%) diets for 20-22 weeks on body weight (A), fat mass (B), lean mass (C), hemoglobin (D), and food intake (E) in male C57BL6/J mice. n=4. (F-G) The 20-ppm iron diet led to a higher glucose clearance capacity indicated by glucose tolerance test (F) and the area under the curve (G). n=4-5. (H-I) The 20-ppm iron diet enhanced insulin sensitivity assessed by insulin tolerance test (H) and the area under the curve (I). n=4-5. (J-K) Respiratory quotient during the dark (black bar) and light (white bar) cycles (J), and the average values for both cycles were shown in panel K. n=4-5. (L-M) The energy expenditure during the dark (black bar) and light (white bar) cycles (L), and the average values for both cycles were shown in panel M. n=4-5. (N-O) Oxygen consumption during the dark (black bar) and light (white bar) cycles (N), and the average values for both cycles were shown in panel O. n=4-5. (P-Q) CO2 production during the dark (black bar) and light (white bar) cycles (P), and the average values for both cycles were shown in panel Q. n=4-5. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 2.

Figure 2.

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Iron alteration in white adipose tissues. (A-B) Serum and liver iron contents indicated no significant changes in systemic iron and iron store in mice on 20-ppm vs 50-ppm iron high fat diets. n=4. (C-D) Iron contents were increased in inguinal and gonadal white adipose tissues (iWAT and gWAT, respectively) in mice on 20-ppm high fat diet. n=3-4. (E-F) Iron contents were comparable in brown adipose tissue (BAT) and skeletal muscle (SM). n=3-5. Tissue iron contents were expressed per mg of wet tissue. (G-K) Western blotting analysis of ferritin (cellular iron status marker) and DMT1 (iron importer), which indicates significant changes in iWAT (H) and gWAT (I) but not in the liver (G), BAT (J) and muscle (K). n=4-6. *, p<0.05; **, p<0.01.

Tissue-specific iron distribution and metabolic fitness.

To determine how iron may affect tissue specific metabolism, we first measured iron distribution in the blood, liver, adipose tissues and skeletal muscle (Figure 2, A-F). Although hemoglobin level was reduced (Figure 1D), iron contents were unchanged in the serum, liver, skeletal muscle (SM), and brown adipose tissue (BAT). Interestingly, inguinal (iWAT) and gonadal (gWAT) white adipose tissues showed higher iron content (Figure 2, C-D) in mice on 20 ppm vs 50 ppm iron HFD. To validate the findings, we analyzed the iron status marker ferritin (Figure 2, G-K), which confirmed the unchanged iron status in the liver, SM and BAT (Figure 2 G, J-K); consistently, the regulator of iron uptake DMT1 remained comparable. Intriguingly, ferritin level (FTL or FTH) was downregulated in iWAT and gWAT from mice on 20 ppm vs 50 ppm iron HFD (Figure 2, H-I) regardless of a higher level of adipose iron (Figure 2, C-D). This reveals the presence of a conditional subcellular “iron deficiency” status, where cytosolic iron is redistributed for mitochondrial biogenesis or to meet an increased iron requirement [23, 24]. In line with the notion of conditional “iron deficiency”, DMT1 was upregulated significantly in iWAT and gWAT (Figure 2, H-I). Of note, we were limited to detect either FTH or FTL (other than in parallel) due to tissue-dependent performance of antibodies.

Moderate iron restriction induces adipose browning.

To characterize the subcellular iron redistribution, we isolated mitochondria from adipose tissues and measured iron content in mitochondrial and cytosolic fractions (Figure 3, A-C). The mitochondrial pellets isolated from equal amount (1 g) of gWAT were greater in mice on 20 ppm vs 50 ppm iron HFD (Figure 3A). Mitochondrial iron content in mice on 20 ppm was 2.2-fold (p<0.01, Figure 3B) of that in mice on 50 ppm, whereas cytosolic iron content was significantly lower in mice on 20 ppm (p<0.05, Figure 3C). Western blotting analysis indicated the upregulation of mitochondrial oxidative phosphorylation (OXPHOS) proteins in iWAT and gWAT (Figure 1, D-E). In addition, mitochondrial uncoupling protein UCP1 was induced by 20 ppm iron diet, concomitant with activation of Pgc1α-Tfam pathway (Figure 3, F-G), the master regulator of mitochondrial biogenesis [25]. Indeed, the marker of mitochondrial mass Tom20 was increased by 20 pp iron diet (Figure 3, F-G). These data suggest that moderate iron restriction induces adipose browning and mitochondrial biogenesis, which favors subcellular iron redistribution from the cytosol to mitochondria.

Figure 3.

Figure 3.

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Iron redistribution during the browning of white adipose tissue. (A) Isolated mitochondrial pellets from 1 g iWAT in mice on 20-ppm vs 50-ppm iron high fat diets. (B-C) Iron content in mitochondrial and cytosolic fractions from iWAT from mice on 20-ppm vs 50-ppm iron high fat diets. n= 4. Iron contents were expressed per mg of protein. (D-E) Western blotting analysis of mitochondrial oxidative phosphorylation proteins indicated an elevation of complexes I, II, III and V in iWAT (D) and gWAT (E) from mice on 20-ppm iron high fat diets. n=4-6. (F-G) Western blotting analysis indicated an elevation of adipose browning marker UCP1, mitochondrial biogenesis markers Pgc1α and Tfam, and mitochondrial content marker TOM20 in iWAT (F) and gWAT (G) from mice on 20-ppm iron high fat diets. n=4-6. *, p<0.05; **, p<0.01; ***, p<0.001.

Moderate iron restriction enhances transcripts of metabolic functions.

To map the metabolic pathways that were affected by 20-ppm iron diet, we performed RNA sequencing and enrichment analysis for mice fed on low fat diet to investigate the sole role of iron restriction with no confounding effects of high fat (Figure 4, A-D). The elevated genes in iWAT and gWAT were shown by heatmaps (Figure 4, A and C), and activated pathways by GO enrichments (Figure 4, B and D). Consistent with mitochondrial enhancement and metabolic improvement described above (Figure 1, Figure 3), we observed the activation of pathways involved in coenzyme oxidoreduction, acetyl-CoA metabolism, carbohydrate and lipid metabolism (Figure 4, B and D). Of note, the metabolic benefit of moderate iron restriction was also observed in mice on low-fat (10%) diet (LFD), concurrent with a mild decline in hemoglobin without causing amenia (Figure 4, E-G). Therefore, moderate iron restriction improves metabolism in mice fed on LFD as on HFD (Figure 1), suggesting the regulatory mechanism prevails under both conditions.

Figure 4.

Figure 4.

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Moderate iron restriction in low fat diet induces genes that regulate mitochondrial and metabolic activities in white adipose tissues. (A-B) RNA sequencing analysis revealed an array of genes upregulated by 20-ppm iron diet (A), most of which were involved in mitochondrial and metabolic regulations in iWAT (B). (C-D) The 20-ppm iron diet upregulated an array of genes (C), most of which were involved in metabolic regulations in gWAT (D). (E-G) In mice on low-fat (10%) diet for 20-22 weeks, 20-ppm iron improved metabolic health assessed by glucose tolerance test (E-F), concomitant with moderate decrease in hemoglobin but no anemia (G), similar to the observation from mice on high fat (60%) diet (Figure 1). n=5. *, p<0.05; **, p<0.01.

Moderate iron restriction improves systemic insulin sensitivity.

GO enrichment also revealed a common biological process in iWAT and gWAT, i.e., cellular response to hormone stimulus (Figure 4, B and D). KEGG pathway analysis highlighted insulin signaling pathway (Figure 5A) among mitochondrial metabolism (e.g., citrate cycle), and carbohydrate and lipid metabolism (e.g., pyruvate metabolism, fatty acid metabolism, AMPK, and PPAR pathway). Consistently, Western blotting analysis demonstrated the improvement of insulin sensitivity in iWAT and gWAT from mice fed on HFD (Figure 5, B and D), assessed by phosphorylated AKT at Thr308 and FoxO1 at Ser253 that reflect the activity of canonical insulin signaling cascade [25, 26]. Interestingly, we observed higher activities of pAKT-pFoxO1 pathway in BAT, skeletal muscle and liver (Figure 5, C, E-F), despite the absence of iron redistribution in these tissues from mice fed on HFD (Figure 2, G, J-K). Thus, iron redistribution in white adipose tissue (Figure 2, H-I) may induce an endocrine factor that improves systemic insulin sensitivity during moderate dietary iron restriction.

Figure 5.

Figure 5.

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Moderate iron restriction enhances systemic insulin sensitivity. (A) RNA sequencing and KEGG pathway analysis of the transcripts upregulated by 20-ppm iron diet in inguinal white adipose tissue, which revealed the activation of insulin signaling pathway among other pathways. (B-F) Western blotting analysis of canonical insulin signaling pathways via pAkt(Thr308)-pFoxO1(Ser253) cascade in inguinal white adipose tissue (B), brown adipose tissue (C), gonadal white adipose tissue (D), skeletal muscle (E), and the liver (F) from mice fed on HFD. n=4-6. *, p<0.05; **, p<0.01; n.s., not significant.

Moderate iron restriction increases H3K4 methylation possibly by mitigating adipose JARID activity.

To explore the regulatory mechanism, we mapped molecular function via GO enrichment analysis, which underscored transcriptional events like histone and DNA binding (Figure 6A). This prompted us to examine histone modifications, the epigenetic mechanism that regulates chromatin structure and gene expression [27]. After extracting high-quality histone proteins from adipose tissues in mice fed on HFD (Figure 6B), we used Multiplex ELISA Assay (EpigenTek) to map histone modifications (including methylation, acetylation, and phosphorylation) and identified H3K4me1 (p=0.0023), H3K4me2 (p=0.0182), H3K4me3 (p=0.0159), and H3K36me2 (p=0.0397) as the pronounced modifications (Figure 6C). Western blotting analysis further verified the changes in H3K4me1, H3K4me2 and H3K4me3 induced by 20-ppm iron diet (Figure 6, D-E). We then measured the activity of iron-dependent H3K4 demethylase [28], JmjC-ARID domain-containing protein (JARID), from mice on 20-pp vs 50-ppm iron diets (Figure 6F). In line with the upregulation of H3K4 methylation, JARID activity was reduced by 28.2% (p<0.05) in 20-ppm iron diet treated mice. Given the observed “iron deficiency” due to iron redistribution into mitochondria (Figure 2, H-I; Figure 3, B-C), moderate dietary iron restriction may increase H3K4 methylation possibly by mitigating iron-dependent JARID activity.

Figure 6.

Figure 6.

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Epigenetic regulation of GDF15 by moderate iron restriction. (A) RNA sequencing and GO enrichment of molecular function analysis suggested that moderate iron restriction regulated DNA and histone binding events. (B) Western blotting analysis of histone extraction and cytosolic fraction for purity (absence of tubulin in histone fraction) and completeness (absence of histone in cytosolic fraction). (C) Multiplex ELISA of histone modifications, which revealed H3K4 methylation as the primary epigenetic changes induced by 20-ppm iron high fat diet. (D-E) Western blotting analysis of H3K4 methylation, which confirmed the results from multiplex ELISA. n=4-6. (F) Measurement of iron-dependent histone demethylase JARID activity. n=3. (G) Chromatin immunoprecipitation and qPCR analysis of the interaction between H3K4me2 and GDF15 gene. n=4. (H) qPCR of GDF15 gene expression in gonadal white adipose tissue from mice on 50-ppm vs 20-ppm high fat diet. n=4. *, p<0.05; **,p<0.01.

Moderate iron restriction induces GDF15 via H3K4 methylation.

Recent studies reveal that GDF15 senses alteration of nutrient status and can enhance insulin action [19, 29, 30]. We asked whether GDF15 was involved in the epigenetic changes and metabolic improvements by 20-ppm iron diet (Figures 1, 4, and 5). To address this question, we first performed chromatin immunoprecipitation with the antibody against H3K4me2 (the most dominant form of H3K4 methylation, Figure 6, D-E) to pull down histone-DNA complex, followed by quantitative PCR (qPCR) to detect GDF15 gene. A 5-fold higher abundance (p<0.01) of GDF15 gene was detected in the histone-DNA complex isolated from 20-ppm iron diet treated mice (Figure 6G). Moreover, qPCR analysis of GDF15 gene expression revealed a 2.0-fold elevation of GDF15 mRNA (p<0.05; Figure 6H), supporting the notion that H3K4 methylation promotes gene expression [31-33]. Our data suggests that moderate iron restriction induces GDF15 gene expression in adipose tissue by increasing H3K4 methylation.

Adipose secretion of GDF15 improves insulin action.

To investigate how the epigenetic mechanism contributes to GDF15 production, we measured GDF15 protein expression by Western blotting (Figure 7, A-E). Both iWAT and gWAT from mice fed on HFD showed significant upregulation of GDF15 by 20-ppm iron diet (Figure 7, A-B). However, GDF15 protein levels were comparable in BAT, liver, and skeletal muscle (Figure 7, C-E), consistent with the observation of no significant iron changes in these tissues (Figure 2). ELISA of serum samples showed a significant elevation of GDF15 (173.1 pg/ml vs 143.6 pg/ml, p<0.01) in mice on 20-ppm vs 50-ppm iron diet (Figure 7F). These findings underscore white adipose tissues as the primary contributor to elevated GDF15 in serum samples. Using siRNA against GDF15, we determined how knockdown of GDF15 in 3T3-L1 adipocytes might affect the benefit of iron restriction (Figure 7G). Iron restriction by deferoxamine (DFO, an iron chelator) significantly increased GDF15 protein expression, in line with the observation of elevated GDF15 in mice (Figure 6, G-H; Figure 7, A-B). DFO enhanced canonical insulin signaling assessed by pFoxO1 at Ser253 [25, 26] (Figure 7H), further recapitulating the phenotype of improved insulin sensitivity in mice (Figure 1, F-I; Figure 5). However, knockdown of GDF15 abolished the beneficial effects of DFO (Figure 7H), supporting the notion that adipose GDF15 mediates the benefits of iron restriction. In addition, pharmacological inhibition of H3K4 methylation by MI-2-2 and WDR5-0103 [34, 35], largely reversed effects of DFO on H3K4 methylation, GDF15 expression, and pFoxO1 (Figure 7I). Together, these data suggest that iron restriction improves metabolism via the H3K4 methylation–GDF15 axis.

Figure 7.

Figure 7.

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Adipose GDF15 improves metabolic health. (A-E) Western blotting analysis of GDF15 in inguinal white adipose tissue (A), gonadal white adipose tissue (B), brown adipose tissue (C), the liver (D), and skeletal muscle (E) from mice fed on HFD, which underlined white adipose tissue as the primary contributor to elevated GDF15. n= 6. (F) ELISA of GDF15 in serum samples. n= 8. (G) Knockdown effects of siRNA against GDF15 in 3T3-L1 adipocytes, assessed by qPCR. n= 3. (H) Effect of DFO and GDF15 knockdown on canonical insulin signaling in 3T3-L1 adipocytes, as assessed by Western blotting of pFoxO1(Ser253). DFO, deferoxamine; GKD, GDF-15 knockdown. n= 6. (I) Effects of H3K4 methyltransferase inhibitors MI-2-2 (10 μM) and WDR5-0103 (100 μM) to mimic JARID activation and of DFO on H3K4 methylation, GDF expression, and pFoxO1(Ser253). n= 6. (J) The epigenetic mechanism by which moderate iron restriction induces adipose browning and enhances metabolic health via GDF15. Adipose browning may form a feedback loop with adipose iron redistribution (into mitochondria), causing subcellular “iron deficiency” that mitigates JARID activity. Moderate iron restriction recapitulates the benefits of therapeutic phlebotomy. Severe iron restriction causes anemia and metabolic derangements. *, p<0.05; **, p<0.01; ***, p<0.001.

DISCUSSION

Our study unravels a novel epigenetic mechanism by which moderate iron restriction enhances adipose browning and metabolic health via GDF15 (Figure 7I). Iron restriction (20-ppm iron diet) triggers adipose iron redistribution into mitochondria, constituting a condition of subcellular “iron deficiency” (Figures 2 - 3). As a result, the iron-dependent demethylase JARID activity is suppressed, which is associated with increased H3K4 methylation and GDF15 expression. Pharmacological inhibition of H3K4 methylation or knockdown of GDF15 abolishes the beneficial effects of iron restriction (Figures 6-7). Notably, severe iron restriction (4-ppm iron diet) caused anemia, underweight, and metabolic derangement (Figure 2s). Our data serves as the first line of evidence that links moderate iron restriction to the nutrient sensor GDF15 via H3K4 methylation. It lends evidence to the working mechanism of therapeutic phlebotomy or iron chelation that is shown to improve metabolism and cause no anemia [10-15].

Iron acts as the cofactor of demethylases that mediate hydroxylation reactions in histone and DNA demethylation [36, 37]. These enzymes include histone demethylase jumonji domain-containing proteins (e.g., JMJD1A and KDM5) and the DNA demethylase ten-eleven translocation (TETs), and they are shown to regulate adipocyte differentiation [37-41]. In addition to governing DNA methylation of target genes such as Pparg and Leptin [39, 42], TET proteins may interact with other epigenetic enzymes (e.g., histone deacetylase, HDAC) to repress genes involved in browning response and metabolic enhancement [43, 44]. Ablation of adipose TET proteins in mice enhances energy metabolism and protects against obesity and insulin resistance [39, 42-44]. Our study shows for the first time that moderate iron restriction mitigates JARID activity, concurrent with elevated H3K4 methylation and GDF15 (Figures 6-7), the nutrient sensor reported to regulate thermogenic responses, food intake, and insulin sensitivity via GDNF family receptor α-like receptor (GFRAL) [17-19]. However, the level of endogenous GDF15 varies largely, accounting for varying effects under physiological (0.1-2 ng/ml) and pathophysiological (up to 10 ng/ml) conditions [19, 45]. For instance, GDF15 may suppress food intake but the effects rely on pharmacological dosage (up to 100 ng/ml); in the physiological range even a 4-fold increase of GDF15 did not reduce food intake [19, 30, 46, 47]. Consistent with this, we did not observe an inhibitory effect on food intake by GDF15 (Figure 1E; Figure 7F). Instead, the mice on 20-ppm iron diet had a greater food intake, presumably due to the higher metabolic rate or energy expenditure (Figure 1, J-Q), the known factor that drives energy intake [48, 49]. Future studies of the iron-H3K4 methylation-GDF15 axis may open a new avenue to develop therapeutic options or dietary interventions to prevent or treat metabolic disorders.

A broad range of iron contents in research diets has been reported in the literature, with 4-8 mg/kg or ppm for iron deficiency diets [5, 6, 50], 35-600 ppm for normal iron diets [5, 6, 11, 51], and 2000-20000 ppm for high iron diets [5, 6, 52, 53]. Given that 35-50 ppm is recommended as normal dietary iron for mice by the National Research Council [20], and that 50-ppm iron is equally effective at supporting conception and pregnancy, and pup development [21], we used 50-ppm diet as the control and 20-ppm diet as a moderate iron restriction diet. Measurement of iron content in serum and other tissues suggests that the mice on 20-ppm diet did not develop iron deficiency (Figure 2, A-F). Hemoglobin showed slight yet significant decrease, but it is still in the normal range (hemoglobin >=13.6 g/dL) [22]. In human participants who receive two phlebotomies at 4-week intervals, hemoglobin drops from 14.3 mg/dl at baseline to 13.3 mg/dl (p<0.05) but did not cause anemia (human hemoglobin < 12 g/dL considered anemic) [14]. In patients who receive three phlebotomies at 2-week intervals, hemoglobin drops from 14.72 mg/dl at baseline to 14.12 mg/dl (p<0.05) but caused no anemia [13]. Thus, our study establishes a valuable model to study moderate iron restriction and metabolic health. Future studies to define “safe” duration and dosage of dietary iron restriction may result in new approaches to preventing or treating metabolic syndrome.

Several questions remain for future studies to address. It is unclear why moderate iron restriction leads to iron redistribution primarily in white adipose tissue. Previous studies show that lipids can act as ligands that sequester and shuffle iron intracellularly, thereby tuning down subcellular labile iron [54-56]. In contrast to other tissues, adipose tissue contains massive lipids, which control iron bioavailability as ligands [54-56] and contribute to unique subcellular (hydrophobic vs hydrophilic) microenvironments governing substrate diffusion and iron-dependent enzyme activity [57, 58]. Emerging evidence suggests that lipid droplets, whose size and number distinguish white (large unilocular droplets) from brown (small multilocular lipid droplets) adipocytes [59, 60], are strongly linked to iron metabolism [61, 62]. These differences might account for a higher sensitivity to moderate iron restriction of white adipose tissue over other tissues. While additional studies are warranted to address how dietary iron restriction initiates iron redistribution in adipose tissues, the browning response and augmented mitochondrial biogenesis can increase iron requirement and promote subcellular “iron deficiency”, constituting a feedback loop to facilitate iron redistribution (Figure 7J) [23, 24]. Moreover, it remains to be defined how other iron storing tissues, such as bone marrow, spleen, and macrophages [63-65], respond to moderate iron restriction. Iron overload weakens bones and increases the risks of osteoporosis and fracture [66]. Severely iron deficient diet (7 ppm) for 52-56 days reduces the populations of most lymphocytes in mouse spleen [67], while acute iron deprivation in macrophage reprograms glucose and lipid metabolism and mitigates inflammation [68]. Future studies of iron metabolism in these tissues will shed light on how adipose tissue may cross talk with bone marrow and spleen to coordinate iron supply for the browning response and mitochondrial biogenesis under the condition of moderate iron restriction.

METHODS

Mice.

C57BL6/J mice from Jackson Laboratory (Strain #:000664) were used for breeding, and the weaned mice (3-week old) were fed on the following diets for 20-22 weeks unless specified otherwise: 50 ppm iron low fat (10%) diet (TD.200508), 50 ppm iron high fat (60%) diet (TD.200510), 20 ppm iron low fat (10%) diet (TD.200507), 20 ppm iron high fat (60%) diet (TD.200509), and iron deficient (4 ppm) high fat (60%) diet (TD.230335) from Teklad (Madison, WI). All the mice were housed in plastic cages on a 12-h (7:00 am – 7:00 pm) light–dark photocycle, with free access to water and diet. All the procedures followed the NIH guideline and were approved by the Institutional Animal Care and Use Committees at the University of Florida.

Indirect calorimetry.

Whole body energy expenditure was analyzed by indirect calorimetry with a TSE PhenoMaster system where mice were housed individually with free access to food and water [69, 70]. Mice were allowed 3 days to acclimate to the new housing conditions before the measurement of O2 consumption (ml kg−1 h−1) and CO2 production (ml kg−1 h−1). Energy expenditure (EE) was calculated using the Weir equation: EE = 3.941 × VO2 + 1.106 × VCO2 and normalized to body weight.

Body composition analyses.

Fat and lean mass were measured on a Bruker Minispec LF90 NMR Analyzer (Bruker Optics, Billerica, MA) as described previously [71].

GTT and ITT.

Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed as described previously [69, 72]. For GTT, the mice were fasted overnight (~14 hours), and the animals were injected intraperitoneally (i.p.) with D-glucose (2 g/kg of body weight), and blood glucose was measured at the indicated time points with Contour Blood Glucose Meters and Test Strips. For ITT, mice were fasted for 4-6 hours, followed by i.p. injection of diluted insulin (1 U/kg Humulin R; Lilly), and blood glucose was measured at indicated time points.

Cell culture and treatment.

3T3-L1 cells (CL-173™, ATCC; RRID: CVCL_0123) were cultured and induced for differentiation as described previously [72, 73]. Insulin was included in differentiation media and after cell differentiation. Lipofectamine® RNAiMAX Reagent (cat # 13778150, ThermoFisher) was used to facilitate the knockdown of GDF15 with siRNAs [72, 74]. H3K4 methyltransferase inhibitors MI-2-2 and WDR5-0103 were used to treat mature adipocytes for 4 days at final concentrations of 10 μM and 100 μM, respectively, as described previously [34, 35].

Iron Assay.

Measuring non-heme iron reflects iron status well in response to both heme iron and non-heme iron intake [21]. Total non-heme iron was analyzed with Iron Assay Kits (Cat # MAK025) from Sigma as described previously [23, 69]. Mitochondria were isolated as described before [75, 76] and processed for iron measurement using the Iron Assay Kits (Cat # MAK025).

qPCR.

RNA was extracted with RNeasy Mini Kits (Qiagen, Germantown, MD, USA) according to the manufacturer’s instruction. The RNA samples were used to synthesize cDNA by reverse transcription PCR using iScript™ cDNA Synthesis Kits (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instruction. Gene expression was analyzed by quantitative real-time PCR on a CFX96 system (Bio-Rad, Hercules, CA, USA). The primers used in this study were 5′- CTG GCA ATG CCT GAA CAA CG −3′ (forward) and 5′- GGT CGG GAC TTG GTT CTG AG-3′ (reverse) for GDF15; 5′- CTC TGG CTC CTA GCA CCA TGA AGA −3′ (forward) and 5′- GTA AAA CGC AGC TCA GTA ACA GTC CG −3′ (reverse) for ACTB as a reference gene.

RNA-Seq.

Total RNA from adipose tissue was prepared with RNeasy Mini Kits (Qiagen), and mRNA was purified from total RNA using poly-T oligo-attached magnetic beads [69, 72]. After library construction, diluting library to 1.5 ng/μl with the preliminary quantitative result by Qubit2.0 and detecting the insert size by Agilent 2100. Q-PCR was used to accurately quantify the library effective concentration (> 2nM), in order to ensure the library quality. Libraries were fed into Illumina machine (Illumina Platform PE150) after pooling according to activity and expected data volume. Raw image data file from high-throughput sequencing was transformed to Sequenced Reads (Raw Reads) by CASAVA base recognition (Base Calling). Raw reads were filtered to remove reads containing adaptors, or >10% bases that could not be determined, or when the Qscore of over 50% bases were <= 5. STAR was used to map the clean reads to the reference genome (Mus MusculusGRCm38/mm10). Differential expression analysis was performed with a DESeq2 R package and enrichment analysis with ClusterProfiler [69, 72].

Histone modification and enzyme analyses.

Histone was extracted using EpiQuik Total Histone Extraction Kits (OP-0006-100, Epigentek), and histone modifications were measured with EpiQuik Histone H3 Modification Multiplex Assay Kits (P-3100-96, Epigentek) according to the manufacturer’s instructions. JARID activity was analyzed with Epigenase JARID Demethylase Activity/Inhibition Assay Kits (P-3082-48, Epigentek) according to the manufacturer’s instructions.

Chromatin immunoprecipitation (ChIP).

ChIP assay was performed as described [77-79]. Briefly, minced adipose tissues were treated with 1% formaldehyde for 10 min, and the crosslinking reactions were stopped by adding glycine to a final concentration of 125 mM. The tissues were then rinsed with cold PBS and homogenized using a Dounce homogenizer. DNA was sheared to 200-700 bp by Bioruptor Sonication (Diagenode) at high power for 30 s ON/30 s OFF, 30 cycles; the DNA-histone complexes were immunoprecipitated with Di-Methyl-Histone H3K4 (9725s) antibody from Cell Signaling Technology (Beverly, MA, USA).

Western blotting.

Tissue and cell lysates were prepared with PLC lysis buffer (30 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM NaPPi, 100 mM NaF, 1 mM Na3VO4) supplemented with protease inhibitor cocktail (Roche), and 1 mM PMSF [69, 72, 73]. Total protein concentrations of the lysates were determined using a DC protein assay kits (Bio-Rad). Antibody (catalog number) information: GAPDH (MA5-15738) antibody was purchased from Pierce (Rockford, IL, USA); FoxO1 (2880s), pAkt-T308 (4056s), Tri-Methyl-Histone H3K4 (9751s), Di-Methyl-Histone H3K4 (9725s), Mono-Methyl-Histone H3K4 (5326s), and α-tubulin (2125s) antibodies from Cell Signaling Technology (Beverly, MA, USA); antibody against DMT1 (bs-3577R-TR) from Bioss Antibodies; antibodies against UCP1 (23673-1-AP), Pgc1a (66369-1-lg), Tfam (22586-1-AP), TOM20 (11802-1-AP), histone-H3 (17168-1-AP), and FTL (10727-1-AP) from Proteintech; Akt (07-372), pFoxo1-S253 (SAB4300094) and FTH (SAB2108662-100UL) antibodies from Sigma-Millipore; OxPhos Rodent WB Antibody Cocktail (45-8099) from ThermoFisher; GDF15 (sc-515675) and HSP90 (SC-13119) antibodies from Santa Cruz. HSP90 or GAPDH was probed as a loading control.

Statistical analysis.

Measurements were duplicated or triplicated, and data were presented as mean ± SEM. Shapiro-Wilk tests were performed to assess normality of data distribution. For normally distributed data, statistical analyses were conducted with parametric methods, i.e., t-tests (comparing two groups) and ANOVA (comparing three or more groups). For non-normally distributed data, non-parametric analyses, i.e., Mann-Whitney U tests (comparing two groups) and Kruskal-Wallis ANOVA (comparing three or more groups) were performed. p < 0.05 was considered statistically significant. When similar p results were produced, parametric method is preferred to non-parametric method because the latter only produces p values and lacks the power of the former in producing mean difference, the size and direction of observed effects, and precision of the estimated difference [80, 81].

Supplementary Material

Supplementary Material

Highlights.

  • Severe iron deficiency causes metabolic disorder

  • Moderate iron restriction improves metabolism and attenuates diet-induced obesity and metabolic derangements

  • Moderate iron restriction increases H3K4 methylation and GDF15 possibly via histone demethylase JARID

  • Pharmacological inhibition of H3K4 methylation or silencing GDF15 reverses metabolic improvements

ACKNOWLEDGEMENTS

This work was supported in part by the AHA (18TPA34230082 and 26RIRA1643854 to Z.C.), USDA/NIFA (7008026 to Z.C), NIDDK (R01DK136722 to F.Y.), NHLBI (R01HL166169 to B.L.), and NIA (R00AG081552 to L.S.).

Declaration of interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Zhiyong Cheng reports financial support was provided by American Heart Association Inc. Zhiyong Cheng reports financial support was provided by National Institute of Food and Agriculture. Feng Yue reports financial support was provided by National Institute of Diabetes and Digestive and Kidney Diseases. Bin Liu reports financial support was provided by National Heart Lung and Blood Institute. Larissa J. Strath reports financial support was provided by National Institute on Aging. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

ABBREVIATIONS

ChIP

chromatin immunoprecipitation

DMT1

divalent Metal Transporter 1

DFO

deferoxamine

ELISA

enzyme-linked immunosorbent assay

EE

Energy expenditure

GDF15

growth differentiation factor 15

GFRAL

GDNF family receptor α-like receptor

GO

gene ontology

GTT

Glucose tolerance test

gWAT

gonadal white adipose tissue

H3K4me

H3K4 methylation

HbA1c

hemoglobin A1c

HDAC

histone deacetylase

HFD

high fat diet

ITT

insulin tolerance test

iWAT

inguinal white adipose tissues

JARID

jumonji domain ARID-containing protein

JMJD1A

Jumonji Domain Containing 1A

KDM5

Lysine demethylase 5

NF-kB

nuclear factor-kappa B

OXPHOS

oxidative phosphorylation

Pgc1α

peroxisome proliferator-activated receptor gamma coactivator 1-α

Tfam

mitochondrial transcription factor A

TET

ten-eleven translocation

Footnotes

DISCLOSURES

No conflicts of interest are declared by the authors.

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DATA AVAILABILITY

All the data will be available on request.

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Associated Data

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Supplementary Materials

Supplementary Material
NIHMS2188579-supplement-Supplementary_Material.docx (353.3KB, docx)

Data Availability Statement

All the data will be available on request.

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