High-Altitude Acclimatization Suppresses Hepcidin Expression During Severe Energy Deficit

Introduction

Steady-state erythropoiesis in healthy human adults produces ∼200 billion new erythrocytes each day, a process that requires 20–25 mg of iron daily (Muckenthaler et al., 2017). The physiological demand for iron increases by an estimated 8 mg iron per day to support the increased rate of erythropoiesis with exposure to hypoxic conditions, assuming the average increase in rate in red blood cell volume across all altitudes is ∼50 mL per week (Sawka et al., 2000). The increased physiological demand for iron is thought to be facilitated, in part, by a suppression in the iron regulatory hormone hepcidin. Hepcidin is a 25-amino acid peptide hormone produced by the liver in response to iron loading, inflammation, and infection (Collins et al., 2008). Hepcidin signals for the internalization and degradation of ferroportin, the primary cellular iron export protein, thereby limiting iron efflux into circulation. Conversely, hepatic hepcidin mRNA is suppressed in mouse models of anemia and hypoxia (Nicolas et al., 2002).

Recent studies have observed significant declines in circulating concentrations of hepcidin after exposure to high altitude (HA) (3400–5400 m) (Piperno et al., 2011; Talbot et al., 2012). Declines in hepcidin are thought to facilitate iron availability for enhanced erythropoiesis with hypoxia. A major hurdle for Warfighters conducting dismounted operations or training in HA regions, particularly for extended periods, is maintaining energy balance. Sojourns to HA are also accompanied by a decrease in appetite (Butterfield, 1999), which is compounded by an increase in resting metabolic rate (Butterfield et al., 1992) and, oftentimes, increased physical activity. Prolonged energy deficit at HA results in greater weight loss with a higher percentage of total body mass loss coming from fat-free mass compared with that at sea level. Recently, we conducted a randomized controlled trial to determine the effects of dietary protein intake during energy deficit at HA on fat-free mass preservation (Berryman et al., 2017). We found that energy deficit during 21 days at 4300 m decreased fat-free mass regardless of protein intake. The objective of this secondary analysis was to determine the combined effects of severe energy deficit and hypoxia on hepcidin and measures of iron status in lowlanders during 20 days of controlled feeding at 4300 m above sea level.

Methods

The study conformed to the principles of the Declaration of Helsinki and was approved by the institutional review board at the U.S. Army Research Institute of Environmental Medicine (USARIEM) and registered at ClinicalTrials.gov as NCT02731066. Human subjects participated in these studies after giving their free and informed voluntary consent. Investigators adhered to DoD Instruction 3216.02 and 32 CFR 219 on the use of human subjects in research.

Participants

Males, age 18–42 years, who were born at altitudes <2100 m and were physically active (programmed physical activity 2–4 day/week), were eligible to volunteer. Exclusion criteria included living or traveled to areas >1200 m for ≥5 days within the past 2 months, metabolic or cardiovascular abnormalities, anemia (hematocrit <38% and hemoglobin <12.5 g/dL), and blood donation within 8 weeks of beginning the study. Twenty-one participants were randomized and 17 participants completed the study and were included in the final analyses (Fig. 1). No participants displayed signs of excessive erythrocytosis during the study period.

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FIG. 1.

Volunteer selection chart. PI, principal investigator; SL, sea level.

Results

Participants' demographics and dietary information after sea level and HA are given in Table 1. As reported previously, total energy deficit at HA was −1849 ± 511 kcal/day, inducing an overall weight loss of 7.9 ± 1.9 kg (Berryman et al., 2017). Dietary iron intake remained constant from sea level to HA. Twenty days at HA resulted in a 12% increase in hematocrit (Fig. 2A; p < 0.01) and a 37% decline in serum hepcidin concentrations (Fig. 2B; p < 0.01) compared to day 1. Serum iron (65.4 ± 16.5 and 104.7 ± 34.2 μg/dL; p < 0.01) and TIBC (317.2 ± 37.2 and 332.5 ± 30.0 μg/dL; p < 0.01) increased, resulting in a 55% increase in transferrin saturation (Fig. 2C; p < 0.01) after 20 days at HA compared to day 1. Ferritin concentrations declined by 18% (Fig. 2D; p = 0.02), sTfR increased by 83% (Fig. 2E; p < 0.01), and the sTfR to log₁₀ ferritin ratio, an index of iron-dependent erythropoietic activity (Punnonen et al., 1997; Beguin, 2003), increased by 99% (Fig. 2F; p < 0.0001) on day 20 compared to day 1. Circulating concentrations of interleukin-6 were not different between days 1 and 20 at HA (11.0 ± 16.6 and 11.9 ± 16.7 pg/ml; p = 0.31).

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FIG. 2.

Changes in hematocrit, hepcidin, and indicators of iron status during energy deficit at HA. Line graphs for each participant (n = 17) and means ± standard deviations are shown for (A) hematocrit and (B) hepcidin, (C) transferrin saturation, (D) ferritin, (E) sTfR, and (F) sTfR/log₁₀ ferritin. An asterisk denotes a significant difference (p < 0.05). HA, high altitude; sTfR, soluble transferrin receptor.

Discussion

In this study, we report suppressed hepcidin concentrations and increased iron availability in lowlanders experiencing severe energy deficit during a 20-day sojourn to 4300 m above sea level. These data suggest that increased erythropoietic activity with HA acclimatization suppresses hepcidin expression to increase iron availability during severe energy deficit.

Dietary iron intake and body iron stores decline with exposure to hypoxia (Richalet et al., 1994; Robach et al., 2004). The former is due to a decrease in overall food intake, which may be explained by decreased appetite, nausea, and vomiting from acute mountain sickness, or a decreased availability of food (Butterfield, 1999). The latter may be explained by a reduction in dietary iron intake and/or iron mobilization to the erythropoietic compartment. The suppression in hepcidin signals for iron release from storage organs including macrophages and enhanced absorption of dietary iron in the intestine (Gassmann and Muckenthaler, 2015). In this study, the decline in ferritin and increase in sTfR suggest a reduction in body iron stores with hypoxia and energy deficit even when dietary iron intake was held constant. The increase in transferrin saturation was a result of increased serum iron and TIBC. In one study, after spending 3 days at 3400 m, participants trekked for a period of 5 days to 5400 m where they remained for 11 days (Piperno et al., 2011). Transferrin saturation decreased compared with that at sea level after 20 days at >3400 m, which was consistent with iron mobilization and transfer to the erythropoietic compartment. These seemingly contradictory findings may be due to the opposing effects of energy deficit and hypoxia on erythropoiesis and iron metabolism. Severe energy deprivation inhibits erythropoiesis (Fried et al., 1957; McCarthy et al., 1959; Donati et al., 1964; Fruhman, 1966), which is accompanied by decreases in erythroid progenitors in the bone marrow to preserve iron stores for vital functions. Regardless, these data indicate a strong overall iron mobilization and further support the concept of increased iron transfer to the erythropoietic compartment during energy deficit at HA.

Recent evidence in humans (Troutt et al., 2012) and rodent models (Mirciov et al., 2018) indicates that food deprivation increases hepcidin production. A time-dependent increase in serum hepcidin was observed in men and women during an 18-, 42-, and 66-hour fast compared with baseline (Troutt et al., 2012). Interestingly, hepcidin declined slightly, but remained elevated compared with baseline, when participants were refed with a small amount of food (500 kcals) in the 24 hours after fasting. Previous studies that have measured circulating concentrations of hepcidin at HA report a >80% reduction in hepcidin with exposure to HA (Piperno et al., 2011; Talbot et al., 2012; Goetze et al., 2013; Lundgrin et al., 2013). Although this study did not test the independent effects of energy deficit and hypoxia on hepcidin, the reduction in hepcidin with HA during energy deficit in this study was only 37% (i.e., >50% less than the other reports). Findings from this study suggest that the magnitude of the reduction in hepcidin at HA may be reduced with energy deficit, but that the increased erythropoietic activity with hypoxia may override the stimulatory effects of energy deficit on hepcidin expression.

The regulation of hepcidin in the context of hypoxia remains unclear. Piperno et al. (2011) observed a strong correlation between serum ferritin and hepcidin at HA and hypothesized that iron or the kinetics of iron use in response to hypoxia may signal for the downregulation in hepcidin (Piperno et al., 2011). Consistent with their findings, the percentage change in hepcidin during HA acclimation and energy deficit in this study was strongly correlated with the change in ferritin (r = 0.894, p < 0.01). However, Talbot et al. (2012) supplied volunteers with intravenous infusion of either 200 mg iron or saline placebo immediately before HA exposure and found that the suppression in hepcidin by hypoxia was too rapid to be due to a reduction in iron availability (Talbot et al., 2012), suggesting that a signal released during erythropoiesis and/or hypoxia may signal for the downregulation in hepcidin.

In this study, HA and energy deficit had no effect on circulating concentrations of interleukin-6, a known inflammatory regulator of hepcidin expression (Nemeth et al., 2004). Erythroferrone (ERFE) is a recently identified hormone encoded by the FAM132B gene that mediates hepcidin suppression during stress erythropoiesis (Kautz et al., 2014). Kautz et al. (2014) demonstrated that erythropoietin stimulates expression of ERFE in the bone marrow through Jak2-Stat5 signaling and that ERFE is secreted by (pro)erythroblasts to suppress hepcidin expression in mouse liver (Kautz et al., 2014). A validated commercially available kit to measure serum ERFE was not available at the time of this study; however, recently, Talbot et al. indicated no relationship between hepcidin and ERFE in HA natives undergoing venesection for chronic mountain sickness (Talbot et al., 2016). Rodents exposed to acute hypoxia (6–15 hours) showed a persistent downregulation in hepcidin and a transient increase in ERFE in the bone marrow (Ravasi et al., 2018), indicating that alternative mediators may be involved in maintaining the downregulation in hepcidin with hypoxia. Further studies are needed to clarify the role of ERFE in hypoxia-induced inhibition of hepcidin.

Collectively, these data indicate that hypoxia is a strong signal for iron mobilization to support iron transfer to the erythropoietic compartment. Importantly, dietary iron and iron recycling from macrophages cannot match the increased requirements for iron at HA. These findings are relevant to military personnel, who often endure sustained periods of energy restriction during training and combat operations in HA regions, for mountaineering expeditions and those trekking to HA, and may be clinically relevant to the hypoxemia and energy restriction observed in critically ill patients.