**3. Hepcidin and Iron Regulation**

Iron status is tightly controlled in the body by the homeostatic regulation of iron movement across the gu<sup>t</sup> and between cells. Homeostasis is essential, not only to encourage iron uptake in times of need, but also prevent iron toxicity and overload. Iron regulation is governed by the master regulatory hormone, hepcidin, which is released from the liver to dictate the availability of iron for biological functions [36]. The primary action of hepcidin is to bind to, and internalize the body's cellular iron export channels, ferroportin, located on the cell surface of macrophages of the reticuloendothelial system, enterocytes in the duodenum and hepatocytes [37]. Hepcidin–ferroportin interactions decrease both the amount of iron that can be absorbed from the diet by duodenal enterocytes, and the amount of iron recycled by macrophages. Through this mechanism, hepcidin is able to regulate transferrin and intracellular iron stores in a homeostatic manner. For instance, in iron-deplete individuals, hepcidin concentrations are reduced as a means of encouraging iron absorption to drive the replenishment of iron stores. However, iron excess stimulates liver hepcidin production, in an attempt to prevent further increases in iron supply [38].

While iron status appears to be a dominant factor in hepcidin regulation, inflammation is also known to impact hepcidin levels, and subsequently, iron balance [39]. The inflammatory cytokine interleukin-6 (IL-6) directly stimulates hepcidin production via an increase in signal transducer and activator of transcription 3 (STAT 3) production, resulting in increased transcription of hepcidin from hepatocytes [40]. This mechanism was highlighted through administration of 30 <sup>μ</sup>g·L−<sup>1</sup> recombinant IL-6, which elicited a 7.5-fold increase in hepcidin concentrations 2 h post-infusion [41]. This outcome was replicated in a study using a 2 ng·kg−<sup>1</sup> body mass (BM) injection of lipopolysaccharide (i.e., stimulating an inflammatory response), in which IL-6 was increased 3 h post-injection, followed by an increase in hepcidin levels 3 h later (i.e., 6 h post-injection, but 3 h post-IL-6 response) [42]. Since exercise is known to be a potent inflammatory stimulus, the relationship between exercise, inflammation, and hepcidin activity has attracted attention. Indeed, IL-6 is released from the skeletal muscle in response to exercise, playing a key role in mediating the acute phase

response [43]. The duration of exercise appears to be the largest determinant of the post-exercise IL-6 response, with increases occurring in a time-dependent exponential manner, peaking immediately post-exercise [44]. Furthermore, exercise intensity and modality can also influence the IL-6 response, with higher intensity and weight-bearing modes (i.e., running vs. cycling) yielding greater increases in post-exercise cytokine levels [43,45]. An early investigation of the link between exercise, IL-6 and hepcidin tracked the time course of changes in these two variables following 60 min of treadmill running (15 min at 75–80% HRpeak, followed by 45 min at 85–90% HRpeak) [46]. Here, a 6.9-fold increase in IL-6 was evident immediately post-exercise, followed by a subsequent peak in hepcidin levels 3–6 h later (5.2-fold increase). This was the first study to demonstrate that increases in hepcidin levels occur subsequent to an exercise-induced inflammatory stimulus. This sequence is important, as it is likely that iron absorption is impaired during the post-exercise period, when hepcidin levels are elevated. This outcome could have negative implications for athletes' iron balance, particularly when performing frequent high-volume training. Within the exercise literature in particular, hepcidin levels have been used as a surrogate marker of iron bioavailability, with interest in strategies that minimize the hepcidin response to exercise.

Multiple regression analysis of both physiological and biochemical markers has shown that the increase in IL-6 concentrations is a small, ye<sup>t</sup> significant contributor to the magnitude of subsequent hepcidin increase at 3 h post-exercise [47]. Interestingly, nutritional manipulation of the magnitude of the IL-6 response to exercise may provide a mechanism to improve iron absorption during the post-exercise period. Factors that influence the release of IL-6 during exercise include the task duration, mode, intensity, training status, and of importance in the context of sports nutrition, muscle glycogen stores [19,48]. Although an increased production of IL-6 has been demonstrated in response to running or cycling for ≥2 h at moderate to high intensities, this response can be attenuated when CHO is consumed throughout the exercise task to maintain blood glucose concentrations and decrease the reliance on/depletion of muscle glycogen stores [19]. However, studies utilizing exercise bouts <2 h in duration have shown CHO supplementation has minimal impact on IL-6 concentrations, unless exercise is commenced with low muscle glycogen stores, in which case the response is augmented [19]. In this low muscle glycogen scenario, CHO ingestion during exercise bouts of 30–90 min in duration can promote the attenuation of the IL-6 response to exercise [49,50]. Given the relationship between IL-6 and hepcidin activity, an increased IL-6 response resulting from training with low CHO availability and/or low muscle glycogen stores, may increase hepcidin levels 3 h post-exercise, which could then negatively impact iron regulation in athlete cohorts. Therefore, strategies that promote CHO availability may help to limit the post-exercise compromise in iron absorption by attenuating exercise-induced inflammation and subsequently minimizing post-exercise hepcidin levels. However, the quantity and timing of CHO intake are important factors in regulating this response.

#### **4. Carbohydrate Availability and Iron Regulation**
