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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Hepcidin, the body's main regulator of systemic iron homeostasis, is upregulated in response to inflammation and is thought to play a role in the manifestation of iron deficiency (ID) observed in obese populations. We determined systemic hepcidin levels and its association with body mass, inflammation, erythropoiesis, and iron status in premenopausal obese and nonobese women (n = 20/group) matched for hemoglobin (Hb). The obese participants also had liver and abdominal visceral and subcutaneous adipose tissue assessed for tissue iron accumulation and hepcidin mRNA expression. Despite similar Hb levels, the obese women had significantly higher serum hepcidin (88.02 vs. 9.70 ng/ml; P < 0.0001) and serum transferrin receptor (sTfR) (P = 0.001) compared to nonobese. In the obese women hepcidin was not correlated with serum iron (r = −0.02), transferrin saturation (Tsat) (r = 0.17) or sTfR (r = −0.12); in the nonobese it was significantly positively correlated with Tsat (r = 0.70) and serum iron (r = 0.58), and inversely with sTfR (r = −0.63). Detectable iron accumulation in the liver and abdominal adipose tissue of the obese women was minimal. Liver hepcidin mRNA expression was ∼700 times greater than adipose tissue production and highly correlated with circulating hepcidin levels (r = 0.61). Serum hepcidin is elevated in obese women despite iron depletion, suggesting that it is responding to inflammation rather than iron status. The source of excess hepcidin appears to be the liver and not adipose tissue. The ID of obesity is predominantly a condition of a true body iron deficit rather than maldistribution of iron due to inflammation. However, these findings suggest inflammation may perpetuate this condition by hepcidin-mediated inhibition of dietary iron absorption.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Iron deficiency (ID) is the most common nutritional deficiency in the United States and has been linked to obesity in adults and children from both industrialized and transition countries (1,2,3,4,5,6,7,8,9). Speculation of the etiology of obesity-associated ID has included inadequate dietary iron intake, increased iron requirements due to increased body size and blood volume, and menstrual irregularities (1,2,3,4,5). Recent studies have ruled out inadequate dietary iron intake as the source of this problem (8,9,10). The latest research has focused on the impact of obesity-associated low-grade inflammation and hepcidin on systemic iron metabolism (6,8,9,11,12).

Hepcidin is a small antimicrobial peptide and the body's main regulator of systemic iron homeostasis (13,14). It is increased by inflammation and elevated body iron levels and decreased by erythropoietic activity and hypoxia (13,14). Hepcidin controls the flux of iron into plasma by post-translational regulation of the body's sole cellular iron exporter, ferroportin-1 (14,15). Acutely high hepcidin inhibits both the absorption of iron from the diet and the release of iron out of storage sites by obliterating ferroportin-1 expression. Elevated hepcidin levels have been associated with the anemia of inflammation, a condition of diminished iron bioavailability and mobilization characterized by hypoferremia and increased cellular iron stores (14,16,17). Recently, mild, increased hepcidin production resulting from mutations in its regulator, TMPRSS6, was described in iron-refractory iron deficiency anemia, a congenital hypochromic, microcytic anemia hallmarked by depleted cellular iron stores, minimal response to oral iron and only partial response to parenteral iron supplementation (18,19,20).

Hepcidin is produced mainly by the liver, but adipose tissue gene expression has been reported with higher mRNA expression in the adipose tissue of obese women compared to lean controls (12). Interestingly, liver hepcidin mRNA expression was shown to correlate with transferrin saturation (Tsat) whereas adipose hepcidin mRNA expression did not and instead was positively correlated with inflammatory parameters, suggesting hepcidin may have tissue-specific regulation. Serum or urinary hepcidin levels were not measured in this study, therefore the contribution of adipose-derived hepcidin to the total circulating levels remains unknown.

The purpose of this study was to determine if serum hepcidin was (i) expressed appropriately in relation to iron status in obese women and if (ii) excess adiposity was associated with inflammation-induced iron sequestration or depleted iron stores. These questions were assessed by measuring: (i) systemic hepcidin concentrations in obese compared to nonobese women matched on hemoglobin (Hb) (Hb levels were used as an indicator of overall iron status), (ii) hepcidin's association with body mass, inflammation, iron status, and erythropoiesis, (iii) hepcidin mRNA expression in the abdominal adipose (visceral and subcutaneous) and hepatic tissue of the obese women to ascertain the relative contribution of hepatic and adipose sources of hepcidin, and (iv) tissue iron accumulation in the obese women's liver and fat depots. We hypothesized that obese women would have increased serum hepcidin compared to nonobese women despite similar serum parameters of iron status (Hb) and that hepcidin levels in the obese women would be positively correlated with adiposity and inflammation and not iron status.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Subject selection

Obese women. Obese women (BMI >37.0 kg/m2) evaluated for bariatric surgery (limited to restrictive procedures: gastric banding or sleeve gastrectomy) were recruited from the University of Illinois at Chicago bariatric surgery clinics between December 2007 and July 2008.

Nonobese women. Hepcidin is decreased in ID through pathways that appear to be independent from the inflammatory regulation of hepcidin (21). Therefore, if hepcidin is responding appropriately to iron status in obese women, with suboptimal iron status, it should be low. As a comparison group, nonobese women (BMI <27.0 kg/m2, waist circumference <88.0 cm), matched to the obese for Hb (range of Hb in obese women: 9.4–14.4 g/dl), and free of inflammation were recruited. Matching the Hb levels of the nonobese to that of the obese was done to disentangle hepcidin's simultaneous regulation in the obese by the opposing influences of inflammation (increasing levels) and ID (reducing levels); hepcidin levels in the inflammation-free nonobese women should be influenced by their iron status only. Restricting the comparison group to the nonobese women matched for Hb allowed us to determine if hepcidin in obese individuals is responding appropriately to their suboptimal iron status or to their inflammatory state. Nonobese women interested in participating attended a prescreening appointment for Hb measurement using a screening hemoglobinometer (STAT-site M β-Hb, Stanbio Laboratories, Boerne, TX). Nonobese women with similar Hb and age (within 5 years) to the obese participants that were free inflammation (i.e., reported they were not being currently treated for an infection or suffer from a chronic inflammatory condition) were eligible for participation.

Women were excluded if they reported significant medical conditions that could influence iron or inflammatory status (i.e., cancer, HIV/AIDS, inflammatory bowel disease, gastrointestinal bleeding, rheumatoid arthritis), had weight loss >3% in the past 3 months, were void of at least one menstrual cycle in the past 12 months, had full or partial hysterectomy, donated blood in the past 3 months, were pregnant or gave birth within the past year, had hemochromatosis or Tsat >45%, or consumed excessive amounts of alcohol (>50 g/day).

Baseline data collection

Participants reported after at least an 8 h overnight fast and were asked not to consume dietary iron supplements, vitamins containing iron or nonsteroidal anti-inflammatory drugs 48 h prior to the research appointment to eliminate possible acute effects on inflammation or hepcidin production. Subjects that reported a cold, flu, or urinary tract infection in the past 2 weeks were asked to reschedule their research appointment for a later date. Obese subjects with scheduled bariatric surgery were also asked to report prior to starting a liquid diet in preparation for surgery typically 10–21 days before the bariatric procedure due to the unknown impact of this type of diet on iron status and hepcidin levels.

The research protocol was approved by the University of Illinois at Chicago Institutional Review Board and participants provided written consent prior to prescreening and/or study entry.

Data collection

Subject characteristics. Demographic, social, and health history data were collected via self-report questionnaire and included information on marital status, race, household income, education level, family size, current health status, reproductive history, menstrual status, and disease prevalence. Social variables collected included alcohol consumption and cigarette use. Disease prevalence was defined as follows: obstructive sleep apnea if Continuous Positive Airway Pressure use, type 2 diabetes if taking blood glucose lowering medications, menstrual irregularities (fibroids, polycystic ovary syndrome) based on self-report, hypertension if taking antihypertensive medications, high cholesterol if taking statins, and osteoarthritis based on self-report.

Anthropometrics. Subjects were weighed to the nearest 0.1 kg in minimal clothing using a digital scale (Tanita BWB-800AS; Tanita, Arlington Heights, IL). Height was measured to the nearest 0.1 mm using a fixed stadiometer (Health O meter, Bridgeview, IL) and waist circumference was measured using a flexible tape, to the nearest 0.1 mm, at the umbilicus.

Dietary and physical activity assessment

To assess total dietary iron and vitamin C intake the validated Block Brief 2000 food frequency questionnaire was utilized (22). The questionnaire is self-administered, contains 70 items and was designed to provide estimates of usual and customary dietary intake over the past 12 months. Physical activity was assessed using the Kaiser Physical Activity Survey (23). This survey is a self-administered questionnaire based on the Baecke Usual Physical Activity Survey (24) and has been validated in women. The last section of the questionnaires asks participants to list three recreational activities they have engaged in most frequently during the past year, along with the frequency and duration of these activities. Activities recorded were assigned a metabolic equivalent value using the standard Compendium of Physical Activities (25) and tallied together resulting metabolic equivalents per week for each individual.

Laboratory assays

All assays were performed on fasted blood samples. For both Hb and hematocrit (Hct) blood was obtained via fingerstick puncture.

Iron, inflammatory, erythropoietic, and metabolic parameters

Hemoglobin was measured by hemoglobinometer (STAT-site M β-Hb; Stanbio Laboratories, Boerne, TX). Hematocrit was measured using a microcapillary reader (International Microcapillary Reader; International Equipment, Needham Heights, MA) following 3 min of centrifugation. Serum iron, total iron-binding capacity (TIBC), Tsat, serum ferritin, high sensitivity C-reactive protein (CRP), glucose and insulin were performed by Specialty Laboratories (Valencia, CA) respectively. Serum iron and TIBC were measured by the ferrozine method. Serum iron <50 µg/dl (normal range 50–170 µg/dl) and TIBC <220 µg/dl (normal range 220–450 µg/dl) indicated ID based on reference ranges provide by Specialty Laboratories. Ferritin was measured by chemiluminescence and values <10 ng/ml (normal range for premenopausal women 10–282 ng/ml) were consistent with ID based on laboratory cutpoints. Tsat was calculated as iron/TIBC × 100; values <20% are consistent with ID based on laboratory cutpoints (26). The analysis of CRP was by immunoturbidity (reference interval <1.0 mg/l), insulin by chemiluminescence (reference range 3.0–28.0 mU/l), and glucose by hexokinase endpoint spectrophotometry (reference range: 74–106 mg/dl). Insulin resistance was determined by the homeostasis model assessment (HOMAIR) according to the following formula: ((glucose (mg/dl)/18) × insulin (mU/l))/22.5 (26). Serum transferrin receptor (sTfR) was measured by Quantikine IVD immunoassay (R&D Systems, Minneapolis, MN). The manufacturer's expected reference range for this assay is 8.7–28.1 nmol/l with a value >28.1 nmol/l indicative of ID per the manufacturer's recommendations. The manufacturer states that sTfR values for African Americans are higher than those of non-African descent. Interleukin-6 (IL-6) was measured by Quantikine quantitative sandwich enzyme immunoassay (R&D Systems) with an expected reference range of 0.447–9.96 pg/ml for this assay. Erythropoietin was measured by immunoassay (mdb biosciences, St Paul, MN) with an expected reference range of 4.3–32.9 mU/ml in serum.

Serum hepcidin

Serum hepcidin was assessed using a competitive enzyme-linked immunosorbent assay developed by Intrinsic Life Sciences (La Jolla, CA). Detailed methods and performance of this assay were recently published (27). The sensitivity for this assay is 0.5 ng/ml and intra-assay coefficient of variation was 5–19% and median interassay coefficient of variation was 12%. For women with normal iron status, the 5–95% range for this assay is 17–286 ng/ml.

Tissue collection (obese only)

During the restrictive bariatric procedure a subset of the obese women consented to a subcutaneous and visceral abdominal adipose surgical biopsy and tru-cut liver biopsy. Fasted serum was collected prior to surgery to assess any significant changes in serum hepcidin, serum iron, and CRP from baseline that could impact tissue iron levels or hepcidin mRNA expression. Adipose and liver tissue samples were fixed in neutral buffered formalin, embedded in paraffin, and four micron sections were stained by hematoxylin and eosin and Perl's iron stain or immediately frozen in liquid nitrogen and held at −80 °C prior to analysis.

Semiquantitative tissue iron accumulation (Perl's Prussian blue staining)

Perl's Prussian blue staining is a classic semiquantitative method to assess iron accumulation within tissues. The degree of iron accumulation in liver and adipose tissue was assessed by an experienced pathologist masked to outcomes using criteria previously published by Rowe et al. (28). Briefly, grade 0 was provided if granules were absent or barely discernible at ×40 objective; grade if barely discernible at ×20; grade if granules seen at ×10; grade if granules seen at ×2; and grade 4+ if granules seen with the naked eye. For the liver, both macrophages and hepatocytes were included in the assessment. For assessment of stainable iron in the adipose tissue, the number of clusters (defined as group of 4–10 macrophages) containing stainable iron was indicated. Grade 0–1+ is considered to be within normal limits for chemically estimated tissue iron accumulation (5–40 µmol/g dry weight), Grade 2+ is suggestive of mild iron overload, whereas 3–4+ represents a significant increase in iron accumulation (130–850 µmol/g dry weight).

RNA isolation

We measured hepcidin mRNA expression in the liver and abdominal adipose tissue of obese women to assess whether hepcidin production by adipose tissue may be a significant contributor to the elevated systemic hepcidin levels. Total RNA was isolated from the liver and adipose (visceral and subcutaneous) tissue. Small pieces of tissue (<100 mg) were homogenized in 1.5 ml of TRIzol reagent (Invitrogen, Carlsbad, CA) which disrupts the cells. The homogenate was centrifuged at 12,000g for 10 min to remove excess fat and cell debris. The cleared homogenate was mixed with chloroform to separate RNA into an aqueous phase. RNA was precipitated using isopropyl alcohol and glycogen, a carrier for nucleic acid precipitation. RNA was washed with 75% ethanol, air-dried and resuspended in RNase-free water.

Quantitative real-time PCR

The complimentary DNA was synthesized from total RNA using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Hepcidin and β-actin mRNA expression were evaluated by quantitative real-time-PCR using iQ SYBR Green Supermix (Bio-Rad). The primers were as follows: human hepcidin, 5′-TTTCCCACAACAGACGGGACAACT-3′ and 5′-GTCTTGCAGCACATCCCACACTTT-3′ and human β-actin 5′-ATCGTGCGTGACATTAAG-3′ and 5′-ATTGCCAATGGTGA TGAC-3′. Amplification was performed at 58 °C for 40 cycles in iCycler Thermal Cycler (Bio-Rad). Data were analyzed using iCycler iQ Optical System Software. The amplification efficiencies of the two products were similar. Hepcidin expression in each sample was normalized to β-actin and results are presented as difference in threshold cycle (ΔCt), where ΔCt hepc = Ct actinCt hepc. With this approach, the lower ΔCt value represents the lower gene expression.

Sample size and statistical analysis

Differences in serum hepcidin between the obese and nonobese women was our main-endpoint and therefore used for determining sample size. Sample size was calculated using the normal approximation for two independent means based on unpublished serum hepcidin means and standard deviation (s.d.) (data provided by coauthor E.N.). The mean (s.d.) for serum hepcidin in those with normal iron status was 91 (±45.7) ng/ml and in the inflamed group 806 (±786) ng/ml. The number of participants required to detect this difference with 80% power at a significance level of 0.05 was 20 per group. Sample size was calculated using the PS: Power and Sample Size program (version 2.1.31; Vanderbilt University Medical Center, Nashville, TN).

Descriptive statistics included mean and race-adjusted geometric mean, s.d., median and interquartile range (IQR) for continuous variables, and frequency for categorical variables. Nonnormally distributed variables were log transformed to achieve normality prior to analysis. Crude comparison between the groups was made using Student's t-test, Wilcoxon rank sum and race-adjusted values using linear models. Dichotomous categorical variables were compared between groups using χ2. Relationships between serum hepcidin and iron status, inflammatory, dietary, and anthropometric variables were assessed using Spearman's test. Variables found to be significant or considered as potential confounders in simple regression were used to develop a best-fit multivariable model to predict log serum hepcidin. Differences in weight, serum hepcidin, serum iron, and CRP between baseline and the day of surgery were assessed in the obese using paired t-tests and Wilcoxon signed-rank. Biochemical, demographic, and anthropometric difference of women refusing liver biopsy were compared using Student's t-test and Wilcoxon rank sum to those consenting to the procedure. Iron accumulation and hepcidin mRNA expression normalized to β-actin from the liver and abdominal adipose tissue from the obese women were assessed descriptively. Spearman correlation between hepcidin mRNA expression and biochemical and anthropometric variables was measured. All P values are two sided and the statistical significance level was set at P = 0.05. All analyses were performed using SAS (version 9.1, 2002; SAS Institute, Cary, NC).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Baseline clinical and biochemical characteristics of the subjects

Forty premenopausal women were recruited (20 obese, 20 nonobese). The groups were similar in age, marital status, education, employment, income, number of offspring, subjective description of menstrual flow, and menstrual duration (data not shown). The majority of women reported light to medium menstrual flow (77.5 %) of 5 days or less in duration (67.5%). Obese women had a higher prevalence of obstructive sleep apnea (n = 6 vs. 0; P = 0.01), osteoarthritis (n = 11 vs. 2; P = 0.002), menstrual irregularities n = 7 vs. 1; P = 0.02), hypertension (n = 8 vs. 1; P = 0.01), hypercholesterolemia (n = 4 vs. 0; P = 0.03), and marginally higher prevalence of type 2 diabetes (n = 3 vs. 0; P = 0.06) compared to the nonobese women (data not shown).

Crude and race-adjusted clinical and biochemical characteristics are presented in Table 1. By design Hb was similar between both groups and expectedly Hct, serum iron, TIBC, and Tsat were also similar although trended lower in the obese. Surprisingly, sTfR was significantly higher in the obese women despite careful matching of the nonobese women for Hb. Dietary iron intake, vitamin C, and notably IL-6 were similar between the women although IL-6 trended higher in the obese. Erythropoietin levels were slightly higher in the obese, possibly because of the higher prevalence of sleep apnea or general hypoxia in this group. As expected, obese women had higher anthropometric measures and CRP. Importantly, obese women also had significantly higher hepcidin levels compared to the nonobese (geometric mean (95% confidence interval): 88.02 ng/ml (53.70–142.03) vs. 9.70 ng/ml (5.24–16.92), P < 0.0001) despite similar serum parameters of iron status including: Hct, serum iron, TIBC, and Tsat and significantly higher sTfR.

Table 1.  Demographic, anthropometric, biochemical, dietary, and physical activity characteristics of obese and nonobese premenopausal women
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Correlations between serum hepcidin and select anthropometric, biochemical, and dietary variables are presented in Table 2. When all women were evaluated, serum hepcidin was positively correlated with BMI, waist circumference, ferritin, CRP, and HOMAIR and not correlated with serum iron. When the obese women were assessed separately, hepcidin correlations were quite different. Hepcidin was positively correlated with both waist circumference (r = 0.51, P = 0.05) and ferritin (r = 0.82, P < 0.0001), but no other significant correlations with iron status markers including serum iron (r = −0.02, P = 0.93), Tsat (r = 0.17, P = 0.49) and sTfR (r = −0.12, P = 0.62) were detected. CRP was not highly correlated with serum hepcidin as anticipated in the obese and IL-6 was only weakly correlated with serum hepcidin. Among the nonobese women, serum hepcidin was positively correlated with ferritin (r = 0.81, P < 0.0001), Tsat (r = 0.70, P = 0.001), and serum iron(r = 0.58, P = 0.01), and inversely with sTfR (r = −0.63, P = 0.004).

Table 2.  Spearman correlations of serum hepcidin with select anthropometric, biochemical, dietary, and physical activity variables analyzed in all subjects and separately by group (obese/nonobese Hb-matched)
inline image

Independent predictors of log serum hepcidin when BMI was entered as a continuous variable in multivariable model building and selection included log ferritin (β = 0.90; P ≤ 0.0001) and log CRP (β = 1.09; P ≤ 0.0001) (adj r2 = 0.71). When BMI was entered as a dichotomized variable (β = 1.93, P ≤ 0.0001), the best model predicting log serum hepcidin also included log ferritin (β = 0.86, P ≤ 0.0001) (adj r2 = 0.77). CRP was no longer a significant predictor of hepcidin likely due to the homogeneity of CRP within each of the groups. Dietary factors, erythropoietin, IL-6, menstrual or disease status were not significant predictors of serum hepcidin in univariate or multivariable analysis. Waist circumference, HOMAIR, metabolic equivalent per week and identifying as African American were significantly associated with serum hepcidin in univariate analysis but lost significance when other variables were included in the model.

Tissue analysis

There were no significant differences in serum hepcidin (103.55 (IQR 107.1) vs. 144.80 (IQR 166.9); P = 0.06), serum iron (42.0 (IQR 24.00) vs. 35.5 (IQR 38.0); P = 0.15), or CRP (11.69 (IQR 9.98) vs. 9.43 (IQR 2.03); P = 0.61) on the day of surgery when compared to baseline in the obese women (n = 20). There were no statistical demographic, anthropometric or biochemical differences between the obese women that consented to a liver biopsy (n = 12) compared to those that declined (n = 8) (data not shown).

Semiquantitative tissue iron accumulation (Perl's Prussian blue staining)

Biochemical analyses indicated that obese women had depleted tissue iron status (elevated sTfR). Semiquantitative analysis (Perl's Prussian blue stain) of the hepatic tissue confirmed that nonheme iron levels were minimal in the obese women that provided samples (n = 12). Only one of the 12 liver samples had visible iron staining but was considered to be within the normal range (0 to +1) (28). We also semiquantitatively evaluated adipose tissue iron accumulation (visceral n = 19; subcutaneous n = 20) based on the hypothesis that local production of hepcidin by adipocytes could lead to iron sequestration in the adipose tissue-associated macrophages, thus further reducing iron bioavailability. Iron staining of subcutaneous and visceral fat indicated very little iron was present in either depot. Only one subcutaneous adipose sample had visible iron staining with a grade of +2, but none of the other 19 subcutaneous or any of the visceral adipose samples had detectable iron levels.

Hepcidin mRNA expression

The obese participants hepcidin mRNA expression, from their subcutaneous (n = 20) and visceral adipose tissue (n = 19), as well liver (n = 12) was normalized to β-actin and reported as ΔCt (Ct actinCt hepcidin) and is presented in Table 3. Specifically, hepcidin gene expression was ∼700-fold greater in the liver compared to either adipose tissue compartment.

Table 3.  Hepcidin mRNA expression in the liver, abdominal visceral and subcutaneous adipose tissue of obese premenopausal womena
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Liver hepcidin mRNA expression was positively correlated with Tsat (r = 0.63; P = 0.05), serum iron (r = 0.86; P = 0.001), and Hb (r = 0.53; P = 0.14) and serum hepcidin (r = 0.61; P = 0.04). Visceral adipose hepcidin mRNA expression was negatively correlated with Tsat (r = −0.52; P = 0.03), serum iron (r = −0.33; P = 0.18) and Hb (r = −0.74; r = 0.007) and weakly with serum hepcidin (r = −0.19; P = 0.43). Subcutaneous adipose tissue was similar to visceral in that hepcidin mRNA expression was negatively correlated with Tsat (r = −0.28; P = 0.23), serum iron (r = −0.25; P = 0.29), and Hb (r = −0.08; P = 0.74) and not correlated with serum hepcidin (r = 0.01; P = 0.95) suggesting that the regulation of hepcidin production differs between the hepatic and adipose tissue.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Obese premenopausal women have significantly higher serum hepcidin levels compared to nonobese women with similar serum parameters of iron status (Hb, Hct, serum iron, Tsat, ferritin), diet and PA patterns. Further, these women suffer from a functional ID as indicated by elevated sTfR and mostly undetectable iron in their abdominal adipose and liver macrophages. Hepcidin decreases with iron depletion and increased erythropoietic activity (13). Thus, based on obese subjects' tissue iron status and hematological parameters, one would expect that serum hepcidin levels would be similar to the nonobese Hb-matched women, if not even lower given their elevated erythropoietic parameters, erythropoietin, and sTfR. When compared to healthy, premenopausal, iron-replete women (n = 25; data provided by co-author E.N.), the concentration of hepcidin (58.30 ng/ml (IQR 88.7); unadjusted value) (27), trended higher in the obese women (103.55 ng/ml (IQR 107.1)) despite their comparatively depleted functional iron status. Although, when compared with women suffering from severe inflammatory conditions (CRP >10 mg/dl), the obese women had remarkably lower serum hepcidin (566 ng/ml (95% confidence interval: 396–989) vs. 103.55 ng/ml (IQR 107.1)) (27).

Although less pronounced, the ID phenotype and hepcidin concentration observed in obesity is similar to iron-refractory iron deficiency anemia (n = 5; serum hepcidin: 133–450 ng/ml) and not the anemia of inflammation (18,19). In persons with the anemia of inflammation, acutely elevated hepcidin is associated with both reticuloendothelial iron sequestration and reduced dietary iron absorption (29). In contrast, mild, persistently elevated hepcidin precipitates a functional ID as suggested by the minimal iron detected in hepatic and adipose reticuloendothelial cells and elevated sTfR observed in the obese women as well as in the patients with iron-refractory iron deficiency anemia. As duodenal enterocytes handle only about 1–2 mg of dietary iron per day, as compared to 20 mg released by the reticuloendothelial system, mildly elevated hepcidin may be sufficient to block dietary iron absorption, but still allow some mobilization of iron from reticuloendothelial cells resulting in depleted functional stores as iron utilization exceeds repletion (30). Corroborating this theory, obese (BMI ≥27 kg/m2) C282Y homozygous patients, have a lower prevalence of hemochromatosis based on Tsat when compared to lean controls (31). Iron overload disorders such as hemochromatosis are the result of disrupted regulation of hepcidin expression by the liver, due to a defect in the Hfe gene, leading to abnormally low hepcidin and increased body iron stores (16). Obesity-associated increase in hepcidin, via the JAK/STAT3 pathway, and consequent reduction in absorption of dietary iron is thought to partially compensate for the defect in the Hfe gene resulting in less severe iron loading in the obese patients (31). In further support, a recent study in obese women and children with suboptimal iron status and elevated sTfR reported decreased dietary absorption of iron-fortified foods compared to lean controls and confirms that iron absorption is diminished in obese individuals despite apparent functional iron depletion (7).

Correlation analysis and linear modeling revealed that hepcidin regulation differed between obese and nonobese women. Central adiposity and inflammation were positively correlated with hepcidin in the obese women whereas iron status parameters were not. In contrast, nonobese subject's hepcidin levels negatively correlated with serum parameters of iron status which resulted in appropriately low hepcidin concentrations. These findings suggest a scenario in which the inflammatory signal counter-regulates the signals related to iron depletion and erythropoiesis. Other studies have analyzed complex regulation of hepcidin by opposing stimuli (i.e., suppression (hypoxia/erythropoiesis) and expression (inflammation/iron overload)) and suggest that hepcidin expression, in the presence of opposing signals, is determined by the strength of the individual stimuli rather than by an absolute hierarchy among signaling pathways (32,33). Therefore, the interplay between inflammation, iron depletion and elevated erythropoietic parameters on hepcidin regulation, to some degree, counter-balance each other resulting in mild but not highly elevated hepcidin observed in the obese women.

Liver but not adipose hepcidin mRNA expression was positively correlated with Tsat in the obese women and corroborates findings of Bekri et al. (12). Feedback regulation to iron via holo-transferrin exists in the liver but similar regulation may not exist in other tissues, such as the adipocyte (34). It has been suggested that the exaggerated fat mass present in obesity could contribute significantly to systemic hepcidin concentrations or it may have a localized effect (i.e., iron sequestration in adipose-associated macrophages) (12). Our findings suggest neither of these two hypotheses is likely. Although mRNA expression does not directly translate to bioactive protein levels, the liver produced 700-fold more hepcidin mRNA compared to the adipose tissue and liver but not adipose hepcidin mRNA expression was positively correlated with serum hepcidin levels. Also, of note, there was little effect related to the localized gene expression in both the adipose and hepatic tissue (i.e., no detectable iron accumulation). These results suggest that most of the circulating hepcidin in the obese subjects is derived from the liver and the adipose tissue, extensive in mass, although likely contributes very little.

Both hepcidin and ferritin are correlated in many conditions (iron-replete healthy individuals, ID anemia, and inflammation) and are regulated by the same stimuli; inflammation and iron (27,35,36,37). Therefore, it was not surprising that both were highly correlated in the obese and nonobese women. Of surprise was the apparent lack of correlation between serum hepcidin and IL-6 as well as CRP in the obese women. Direct IL-6 stimulation has been shown to significantly increase hepcidin via the JAK/STAT3 signaling pathway (35,38). The small sample size and homogeneity of both markers in the obese, likely limited the power to detect differences in IL-6 as well as CRP between the groups and any associations with serum hepcidin. Another possibility is that hepcidin may be regulated by other adipokines commonly elevated in obesity including leptin. Increased levels of leptin have been shown to stimulate hepcidin mRNA production via the JAK/STAT3 pathway in a similar fashion as IL-6 (39). Therefore, leptin may be part of the axis that links obesity, inflammation, and hepcidin with depleted iron stores. The relationship between leptin and hepcidin expression warrants further investigation in obese populations.

Our study has several limitations. First, the study was cross-sectional so causal conclusions cannot be made. Although we are able to assess hepcidin mRNA gene expression we were not able to quantify how this directly relates to bioactive expression of the protein and contribution of such from each tissue or if the mature peptide is identical from both tissues or were we able to compare to our findings with tissue from nonobese individuals, warranting further investigation. Our assessment of iron stores in adipose and hepatic tissue using Perl's staining does not quantify the amount of iron in each tissue. If iron accumulation is less than what can be observed under microscopy by a trained pathologist, then iron accumulation is scored as zero although iron may still be present within the tissue therefore, a quantitative approach is necessary to confirm our findings. Finally, although not assessed, the iron regulatory element/iron regulatory protein system can be altered not only by iron status but also inflammation and may play a role in translational and post-transcriptional regulation of proteins involved in iron uptake, storage, and release in obese individuals and should be assessed in future investigations.

Conclusion

Obesity is associated with iron depletion and elevated levels of serum hepcidin. Further, their reduced iron status does not appear to be due to inflammation-induced iron sequestration within the liver, visceral, or subcutaneous adipose tissue. Our findings suggest obese women have a hepcidin-induced ID in which dietary iron is inefficiently absorbed by the body, similar to the recently described genetic condition iron-refractory iron deficiency anemia and corroborated by the decreased dietary iron absorption observed in obese, mildly iron deficient, women and children. Implications of these findings are significant and could impact iron supplementation and metabolism research in obese populations. Further studies with greater number of racially diverse participants and exploration of the tissue-specific interaction between hepcidin and ferroportin-1 at key iron acquisition and storage sites and quantification of tissue iron content in obese populations are warranted.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

We acknowledge Carlos Galvani, Subhashini Ayloo, and Joseph Vitello for help in obtaining the tissue samples. We also recognize Karin Testa, Melissa Gove, and Zorayda Ortiz in aiding with data collection and analysis. The authors' responsibilities were as follows: L.T.H., E.N., and C.B.: design of the study; L.T.H., S.F., G.Z. and C.B.: data analysis; L.T.H., C.B., E.N., and S.F.: interpretation of the data; L.T.H.: writing of the manuscript draft; C.B., E.N., G.Z., S.F. and A.H.: critical revision of the manuscript. This project was internally funded by the Department of Kinesiology and Nutrition at the University of Illinois at Chicago. E.N. is affiliated with Intrinsic LifeScienes the company that assessed the serum samples for hepcidin.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES