Iron is important for oxygen transport and ATP synthesis. If these processes are impaired by iron deficiency, cellular adaptations occur, such as an increased glucose dependence, in response to that deficiency [1, 2]. The 5’AMP-activated protein kinase (AMPK) has been characterized as a major cellular energy sensor , which may mediate some of these adaptations. AMPK is activated in response to energy challenges such as hypoxia, muscle contraction, and hypoglycemia. Therefore, because AMPK is central to how cells respond to changes in the energy status of the cell and iron homeostasis is critical for the transduction of energy within the cell, we set out to investigate the effects of iron deficiency on AMPK activation and signaling. Ultimately we believe that this information may help elucidate why specific cellular responses occur with changes in cellular iron status.
Iron deficiency is the most common worldwide nutrient deficiency. Of the world’s total population, 24.8% of individuals are anemic . Anemia occurs at all stages of the life cycle, in both developing and developed countries, being most prevalent in pregnant women and young children (41.8% and 47.4% respectively) . Iron deficiency is a metabolic stress because it compromises both the capacity for oxygen supply to tissues (anemia), as well as the capacity to utilize oxygen due to impairment of mitochondrial capacity . This type of energetic stress, due to either decreased oxygen supply or utilization, has been shown to cause an increase in AMPK activation .
AMPK is a cellular energy sensor that when activated, stimulates catabolic processes that increase ATP synthesis, and concurrently inhibits anabolic processes that consume ATP . Nutritional or environmental stress, such as hypoglycemia, hypoxia, and/or muscle contraction, lead to an increase in the AMP:ATP ratio . The function of the enzyme is altered by the interaction of the AMPK subunits as conformational changes occur. AMPK is a heterotrimer consisting of one alpha catalytic subunit and two regulatory subunits, beta and gamma , and multiple isoforms of all subunits have been identified (α1, α2, β1 ,β2, γ1, γ2, γ3) [10, 11]. AMPK complexes containing the α2 isoform are more sensitive to changes in AMP concentration than are complexes containing α1 . Furthermore each isoform of the α subunit affects different downstream signaling pathways. For example, the α1 subunit is more important in the inhibition of protein synthesis via the mTOR pathway [13, 14]. The activation of AMPK is largely determined by phosphorylation of Thr172 on the α subunit, which causes a greater than 20-fold increase in activity . This is primarily done by the predominant AMPK kinase in skeletal muscle, LKB1 [15, 16], and is enhanced when the AMP:ATP ratio is high by nucleotide binding to the γ subunit of AMPK . Binding of AMP to the γ subunit increases activation of AMPK by up to fivefold, but also makes AMPK a poorer substrate for the phosphatase and increases phosphorylation by LKB1 (a net increase in activity of >1000-fold) [18, 19]. Conversely, when the AMP:ATP ratio is low, the nucleotide binding sites on γ are occupied by ATP, eliminating inhibition of the phosphatase, decreasing net enzymatic activity .
Exercise is a metabolic stress on the cell, which has been shown to increase the activation of AMPK [21, 22]. Muscle contraction during exercise increases the AMP:ATP ratio because excitation-contraction coupling depends on the hydrolysis of ATP to ADP as its source of energy. Myokinase catalyzes a reaction that transfers a phoshoryl group from one ADP to another, which regenerates one ATP, and forms one AMP (2 ADP → ATP + AMP). This reaction is important for limiting the increase in ADP when the rate of ATP hydrolysis is high, and thus results in an increase in the AMP:ATP ratio [3, 23, 24]. The concerted effects of iron deficiency and muscle contraction on AMPK activation however are still unknown. A muscle cell that is metabolically stressed due to iron deficiency may be even more adversely affected by exercise than an iron sufficient cell. This increased stress likely has consequences on AMPK activation and signaling.
The effects of hypoxia on AMPK activation have been documented , as well as the adverse effects of iron deficiency on mitochondrial enzyme content [5, 26, 27], and AMPK phosphorylation due to iron deficiency . The objective of this study was to examine: 1) the extent to which chronic AMPK activation occurs due to iron deficiency, 2) how AMPK activation and signaling due to muscle stimulation is affected during iron deficiency, and 3) the effects of iron deficiency on the AMPK subunit composition in skeletal muscle.