Iron metabolism and ferroptosis in type 2 diabetes mellitus and complications: mechanisms and therapeutic opportunities

1Iron is an important trace element for living organisms as it participates in a range of metabolic processes, such as oxygen transport, energy metabolism, nucleotide synthesis, and electron transport. Although vital, excessive amounts of iron can be toxic, therefore, its concentration needs to be maintained within an ideal range. Iron homeostasis is regulated and maintained by iron metabolism. Thus, iron metabolic homeostasis is required for the optimal functioning of fundamental physiological processes. Iron homeostasis in humans is regulated by balancing iron uptake with intracellular utilization and storage. Dietary iron is absorbed by duodenal enterocytes (section 3.1) and binds to transferrin in the plasma. Transferrin limits the production of toxic free radicals and is responsible for ferric-ion delivery into cells. The iron homeostasis system maintains transferrin saturation at physiological levels. During iron metabolism, less than 10% of the iron demand is met by intestinal absorption, and the remaining iron is exported by ferroportin. Ferroportin is regulated by hepcidin, which is a peptide hormone and is often secreted by hepatocytes. Abnormal iron metabolism mainly presents as iron deficiency or overload, which triggers multiple pathological changes such as ferroptosis. Ferroptosis is an iron-dependent form of non-apoptotic cell death, characterized by iron overload and lipid hydroperoxides accumulation.

2Numerous studies have shown that ferroptosis plays an important role in the development and progression of type 2 diabetes mellitus (T2DM) and complications. T2DM is a serious global health concern. Physical inactivity and an unhealthy diet are the major T2DM risk factors, and an increasing disease prevalence is observed in children and younger adults. High levels of ferroptosis, mediated by multiple metabolic pathways and signals, can lead to insulin resistance (IR), abnormal metabolism in the liver and fat, and neurological and vascular diseases. Human blood glucose homeostasis is primarily regulated by insulin and glucagon, which promote glycogen synthesis and breakdown, respectively. It has been shown that iron metabolism is involved in different processes of human glucose metabolism such as insulin secretion, liver metabolism, and fat metabolism, and maintains blood glucose homeostasis in multiple organs and tissues.

3Recently, scholars gradually found a relationship between iron metabolism and glucose homeostasis. However, studies were largely limited to animal models, and only a few clinical trials were conducted. In this review, we discuss the iron metabolism processes that are involved in glucose homeostasis, explore potential drug targets related to ferroptosis in T2DM and its complications, and list drugs or small molecules that may inhibit ferroptosis by targeting T2DM and its complications. This review provides a basis for a potential treatment approach and its potential clinical applications.

4Ferroptosis is a newly recognized type of iron-dependent cell death, characterized by iron overload and lipid peroxidation accumulation. In 2003, Dolma et al. first identified the compound erastin, which exhibited selective lethality against cancer cells expressing RAS, but it was noted that cells died in a manner different from that typically observed with known programmed cell death. With the continuous development of this research, Dixon et al. first proposed the concept of ferroptosis, based on its distinct morphological characteristics and function in 2012. Ferroptosis was defined as an iron-dependent form of regulated cell death that involves the iron-catalyzed accumulation of lethal lipid peroxides. Under physiological and pathological conditions, cell death is an inevitable and important function in biological processes and marks the end of cell life. Cells undergoing ferroptosis have different morphological and metabolic characteristics from other known forms of cell death (such as apoptosis, necrotizing apoptosis, and pyroptosis). Morphologically, ferroptosis mainly occurs in cells, presented as decreased mitochondrial volume, decreased or no mitochondrial cristae, and increased bilayer membrane density; however, the nuclear size remains unchanged. Ferroptosis is regulated by many aspects of iron metabolism, including iron absorption, transport, storage, and utilization (section 4.1). In addition, ferroptosis-inducing factors can affect different pathways of glutathione peroxidase directly or indirectly, resulting in a decreased capacity of antioxidants and accumulation of lipid reactive oxygen species (ROS) in cells, culminating in oxidative cell death. Thus, ferrotropis regulation is closely related to the metabolism of iron, lipids, amino acids, and glutathione. Evidence shows that Abnormal ferroptosis is closely related to the occurrence and progression of various diseases, including metabolic diseases, such as T2DM. Over the past decade, an increasing number of studies have supported the view that ferroptosis plays an important pathophysiological role in the occurrence and development of T2DM and its complications.

5In the human being, iron binds to transferrin in the plasma. Transferrin limits the production of free radicals and is the main carrier of iron to cells; the transferrin receptor (TfR) binds to iron to form a trivalent iron complex (Tf-Fe3+) and is transported to a tissue cell that contains a transferrin receptor. Presently, the evaluation of plasma ferritin concentration is a clinically useful method for measuring iron storage. In the human body, it is a sign of iron overload that the plasma transferrin is greater than 45%. And iron overload is a known risk factor for T2DM. The initial description of IR may be in patients with hereditary hemochromatosis (HH). The mechanism of HH is iron deposition in pancreas β cells, and HH induces cell death which leads to diabetes. A systematic review showed that the TfR to ferritin ratio was negatively related to the risk of T2DM and that plasma transferrin may be related to diabetes development directly or indirectly. A cohort study showed that higher plasma serum ferritin levels were significantly associated with an increased risk of T2DM. These results support iron intake and storage as an indicator of T2DM, which could potentially allow for early diagnosis in clinical practice. Due to the relationship between iron metabolism and glucose homeostasis, maintaining normal iron metabolism is a key factor in maintaining blood glucose stability. Below, we discuss the key processes of iron metabolism in maintaining blood glucose homeostasis, in different tissues under physiological and pathological conditions (Fig. 1).

6The main source of iron is diet. Upon intake, iron is primarily absorbed by intestinal cells in the form of free Fe2+ or heme iron. Free Fe2+ in the intestine is absorbed by intestinal cells through divergent metal transporter 1 (DMT-1). Heme iron is transported to the intestinal cells through heme carrier protein 1 (HCP-1). In the basolateral membrane of the intestinal epithelium, Fe2+ is released into the blood capillary through ferroportin, oxidized by hephaestin, combined with transferrin in the circulation, and transported to various organs and tissues.

7Iron plays an important role in insulin secretion function in pancreas β cells. Tf-Fe3+ is absorbed into pancreas β cells through DMT-1. The pancreas β cells strictly control iron homeostasis, to avoid excessive harmful free iron. Consequently, Fe2+ is reserved in the labile iron pool (LIP), where iron is sequestered by ferritin (a unique cytoplasmic iron storage protein). Fe2+ is bound in ferritin for the synthesis of iron-dependent proteins in the cytoplasm or mitochondria. In extracellular, the pancreas β cells release hepcidin, which binds transferrin and induces their internalized. Studies have shown that transferrin mediates a positive feedback mechanism for iron regulation in the process of glucose-stimulated insulin secretion. Fe2+ in the Labile iron pool (LIP) is involved in insulin secretion, via three pathways. Although iron can be found in almost all intracellular organelles, iron is predominantly consumed by mitochondria which is the primary source of cellular iron metabolism. Synthesis of heme and Fe-S clusters, used for electron transport proteins, occurs in the mitochondria. Under high-glucose stimulation, glucose enters the pancreas β cells via glucose transporter 2 (GLUT-2) and undergoes glycolysis before entering the mitochondria, which leads to increased ATP production. Iron exchange in the mitochondria is mediated by DMT-1 and classical mitoferrins (Mfrn) 1 and 2, which are incorporated into the electron transport chain and produce ATP under the stimulation of glucose. An increase in the ATP to ADP ratio triggers insulin secretion. In addition, Fe2+ promotes ROS production via the Fenton reaction, which is regarded as an amplified signal for insulin secretion. Recent research has shown that Fe2+ participates in Fe-S cluster biosynthesis. The Fe-S cluster promotes the Cdkal1 catalytic metabolism of t6A37 in tRNALysUUU to ms2t6A37 and enables the normal processing of proinsulin into insulin. The Fe-S cluster is an iron mitochondrial chaperone, expressed in pancreas β cells and stimulated by hyperglycaemic disease. Iron is a cofactor of several enzymes and an important component of the Fe-S cluster, participating in insulin secretion as well as in the proliferation and differentiation of β cells. Proinsulin translation in pancreas β cells requires the activity of the Fe-S cluster enzyme, CDKAL1. CDKAL1 dysfunction leads to lysine codon misreading in proinsulin and impairs proinsulin processing, thereby reducing insulin concentration and secretion. A high-glucose environment will lead to an increase in extracellular hepcidin concentration and inhibit the excretion of intracellular Fe2+ through ferroportin. It was found that upon glucose stimulation, islet tissue from iron-deficient mice showed impaired insulin release. In glucose depleted environment, human pancreas β cells upregulate the expression of the TfR. However, excess TfR may be toxic due to excessive activation of the oxidation pathway and ROS accumulation. The redox-active iron form (Fe2+) oxidizes lipids, in the Fenton reaction, which results in the production of a large amount of ROS, further oxidation of DNA and proteins are mediated by ROS, which results in insulin synthesis and secretion reduction, and ultimately apoptosis (Fig. 2).

8Iron is an important regulator of energy metabolism, primarily in adipose tissue. Adipose tissue has specific dynamic characteristics that are helpful in regulating the steady state of carbohydrate and lipid metabolism in the human body. Adipose tissue regulates metabolism by responding to signaling molecules, such as adipokines produced by other metabolic tissues (for example by crosstalk between the liver and skeletal muscle). Mitochondrial dysfunction in the adipose tissue is a key determinant of the etiology of type 2 diabetes. Iron deficiency and iron overload are considered important causes of chronic metabolic diseases (such as T2DM or obesity). Studies have shown that the expression of ACO1 is positively related to adipogenic markers in adipose tissue. Although the mechanism of ACO1 and transferrin affecting adipose tissue metabolism is unclear, ACO1 gene expression is significantly related to gene expression of proliferator-activated receptor-gamma coactivator 1-beta (PGC-1β), which is critical to the mitochondrial function of adipose tissue. Therefore, further research is required to clarify the relationship between the mitochondrial oxidation capacity of adipocytes and iron regulation. Iron is the key regulator of mitochondrial biogenesis. As an important component of the Fe-S cluster, iron is necessary for mitochondrial oxidation regulation. It was found that reducing Mfrn1/2 in mitochondria reduced the mitochondrial iron content, oxygen consumption rate, and ATP level in fat cells, which lead to a reduction in lipogenic gene expression and lipid synthesis during lipogenic differentiation. Studies have shown that mice fed with rich iron diets showed upregulation of IR-related adipokines. In addition, the intervention of rat adipocytes with excessive iron leads to reduced glucose transport, after insulin stimulation. Adipocytes are regulated by various cytokines, such as leptin and adiponectin. Studies have shown that leptin and adiponectin levels are reduced in the adipocytes of mice fed a high-iron diet. Clinical experiments have shown that patients with T2DM have higher transferrin and lower adiponectin levels than healthy individuals. Therefore, abnormal iron homeostasis leads to changes in the levels of various fat factors, eventually leading to lipid metabolism disorders and IR.

9The relationships among iron metabolism, T2DM, liver function, and liver injury are complex. Hepatocytes play a dual role in iron metabolism: they serve as the main site of iron storage and regulate blood iron content by secreting regulatory hormones, such as hepcidin, which controls plasma iron content by binding to transferrin in intestinal epithelial cells and iron circulating macrophages. The binding of transferrin to hepcidin triggers transferrin degradation, thereby reducing transferrin levels. Hepcidin expression is primarily regulated by the BMP-SMAD signaling pathway. In addition, hepatocyte metabolism is regulated by iron. Studies have shown that the iron-sequestering ferritin H chain (FTH) is synthesized in hepatocytes to limit iron-induced hepatic glucose-6-phosphatase (G6Pase) expression and oxidative inhibition. FTH maintains endogenous glucose production, through hepatic gluconeogenesis, which is necessary for hypoglycemic prevention.

10Studies have shown that iron overload causes IR, which is the risk factor in T2DM and non-alcoholic fatty liver disease (NAFLD). In multiple models of IR, researchers have found that iron overload leads to the development IR. It was shown that hepatic gluconeogenesis is increased in mouse models of hereditary haemochromatosis. In db/db mice, iron overload aggravates IR and increases hepatic gluconeogenesis. In hypoxia and iron deficiency mouse models, iron restriction caused hypoglycemic, in part due to reduced hepatic gluconeogenesis, possibly due to the activation of the hypoxia-sensing pathway. In the context of the functional interplay between iron metabolism and liver gluconeogenesis, studies have shown that iron can alter the circadian rhythm of hepatic glucose production and affects liver gluconeogenesis (section 2.6). Inappropriate hepcidin synthesis has been shown to play a role in the pathogenesis of T2DM and its complications. Insufficient hepcidin expression results in iron overload, which triggers ROS synthesis which in turn plays a major role in the pathogenesis of β cell exhaustion and IR-mediated T2DM. Increased hepcidin expression leads to increased intracellular iron sequestration and is associated with T2DM complications.

11Studies have shown that progressive iron storage and T2DM development, in obese patients, causes aging, and affect the brain microstructure and function. Iron metabolism also influences the composition of the gut microbiota, which is also known to affect cognition via the gut-brain axis. Gut microbiota has emerged as an important risk factor for T2DM and obesity. Therefore, these results suggest a link between iron metabolism, the composition of gut microbiota, and the development of T2DM.

12Luconeogenesis is usually suppressed during feeding periods and enhanced during fasting. Circadian rhythm disruption is associated with T2DM, both in experimental animal models and humans, and researchers have found an elevated risk of T2DM in night-shift workers compared to normal individuals. Based on the above findings, researchers have discovered that iron could alter the circadian rhythms of hepatic glucogenesis. Dietary iron regulates circadian glucose metabolism through heme-mediated regulation of the interaction of nuclear receptor subfamily 1 group d member 1 (Rev-Erbα) with its co-suppressor, nuclear receptor corepressor 1 (NCOR). In addition, it was found that iron participates in the regulation of β cell function, mediated by biological clock-based mechanisms driven by HIF-1α. Glucose metabolism and insulin release in β cells are controlled by this mechanism, mainly based on the HIF-1α, which can bind promoter regions of clock genes and control their transcription. Some iron-related genes are transcriptionally regulated by clock genes, which regulate circadian rhythms and iron homeostasis.

13Ferroptosis is a newly discovered process of non-apoptotic cell death that is dependent on excess cellular iron uptake. Ferroptosis is associated with reduced mitochondrial volume, and unlike known programmed death pathways, it is not associated with organelle swelling, chromatin condensation, or autophagy. Instead, it is characterized by iron accumulation, lipid peroxidation, and reduced glutathione peroxidase 4 (Gpx4) expression. In vivo studies have found a potential association between excessive iron storage and T2DM. This partly reveals a correlation between iron and T2DM, which is closely associated with the development of IR. Therefore, the prevailing view is that the higher the iron storage, the higher the risk of developing T2DM. However, this has not yet been effectively demonstrated. Reducing iron storage levels in vivo, has resulted in improved insulin secretion and peripheral tissue insulin sensitivity, which lead to better control of blood glucose and T2DM condition improvement. Herein, we discuss the ferroptosis pathways and molecules involved in the development of T2DM and its complications. The link between ferroptosis and the development of T2DM and its complications has not yet been fully elucidated.

14It is well known that one of the main antioxidant protective enzymes in cells is GPx4, whose lipid peroxide reduction activity plays a crucial role in protecting cells from iron-induced damage and death. Several studies have shown that pancreatic β cells are predisposed to ferroptosis (Fig. 3). Study was shown that pancreatic β cells express low levels of antioxidant enzymes, such as superoxide dismutase (SOD), glutathione (GSH) peroxidase and catalase. Thus, pancreatic β cells are susceptible to oxidative stress. In pancreatic β cells, the Fe2+ in the labile iron pool (LIP) promotes ROS synthesis, via the Fenton reaction. ROS accumulation triggers several ferroptosis. It was shown that the glucose-stimulated insulin secretion (GSIS) capacity of pancreatic β cells was significantly reduced when treated with erastin, in vitro. In contrast, pretreatment with the ferroptosis inhibitors, Fer-1 or DFO, reversed the damage caused by GSIS. External factors (e.g. chronic arsenic exposure) cause mitochondrial damage and produce excess mitochondrial ROS (MtROS), which leads to MtROS-dependent autophagy and ferroptosis, resulting in an increase in intracellular iron. This results in the increased production of Fe2+ in pancreatic β cells, and impaired insulin secretion. It was experimentally verified that MtROS-mediated pathway blocking promotes pancreatic β cell insulin secretion. An Abnormal Fe-S cluster content in cells can easily lead to ferroptosis. The Fe-S cluster regulates iron homeostasis in mitochondria. Iron accumulation in the mitochondria causes a lack of Fe-S clusters, which leads to increased ROS levels in the mitochondria, followed by ferroptosis due to lipid peroxide accumulation.

15Theoretically, increased iron availability may contribute to diabetic macrovascular disease improvement because free iron has adverse effects on the endothelium and accelerates the development of atherosclerosis. In animal experiments, mice fed with an iron-deficient diet had a reduced incidence of atherosclerotic lesions, and iron overload led to a reduction in atherosclerosis. Inhibition of iron-catalyzed oxidative reactions by Deferoxamine (DFX) restores the dilation of the coronary microcirculation and a normal match between myocardial metabolic demand and coronary blood flow in patients with T2DM.

16Early development and accelerated course of diabetic nephropathy have been observed in patients with thalassemia, which is a recognized condition of iron overload. Increased levels of iron in lysosomal proximal renal tubules have been observed in patients with diabetic nephropathy. This observation is related to mutations in HH that appear to predict DN development of diabetic nephropathy. Recent studies suggest that ferroptosis may enhance diabetes nephropathy and impair the renal tubule in diabetes models, via the HIF-1α/HO-1 pathway. Iron increases diabetic kidney injury by increasing oxidative/nitrifying stress and decreasing antioxidant capacity. In addition, iron may be a potential cofactor in diabetic nephropathy, and strict control of iron is therefore important in the diabetic state.

17Experimental studies have shown that DFR administration restores motor and sensory nerve conduction velocity, and improves neural blood flow. Several studies have shown a direct beneficial effect of reducing blood glucose and HbA1c levels, after treatment with high-iron concentrations. The number of pro-inflammatory M1 macrophages was reduced in the neural sections and the number of anti-inflammatory M2 macrophages was increased in db/db mice (fed a high-concentration iron diet). These results confirm and extend the finding in STZ diabetic rats, suggesting that dietary non-iron supplements may partially prevent the development of peripheral diabetic neuropathy (PDN).

18Ferroptosis is one of the reasons for the onset of T2DM and its complications; thus, ferroptosis is a very promising therapeutic target for the treatment and prevention of T2DM and metabolic diseases. In this section, we summarize some molecules that can inhibit ferroptosis and discuss the use of these molecules in the different metabolic pathways of ferroptosis (Table 1).

19Metformin, a biguanide, has been used as the first-line treatment for T2DM for several decades. It has been reported to regulate cellular energy homeostasis by inducing the AMPK signaling pathways. Its basic pharmacological effects include hepatic gluconeogenesis inhibition, glucose uptake promotion, and insulin sensitivity promotion in peripheral tissues. The LKB1/AMPK signaling pathway plays an important role in glucose homeostasis. A previous study showed that LKB1 and its downstream AMP-activated protein kinase (AMPK) blocked ferroptosis by inhibiting the phosphorylation of both acetyl-CoA carboxylase 1 (ACC1) and FAS. Metformin protects cells by activating the AMPK pathway, regulating metabolism, and protecting them from degradation and pathogenic changes at the molecular level. Therefore, we propose that the ability of metformin to improve T2DM is associated with the inhibition of ferroptosis and the reduction of IR. In addition, supplementation of vascular smooth muscle cells (VSMCs) with metformin can enhance the antioxidant capacity of VSMCs, inhibit ferroptosis, and attenuate hyperlipidemia-related vascular calcification through the activation of Nrf2 signaling.

20Quercetin, one of the most widely distributed flavonoids, has been reported to have a large number of attractive pharmacological efficacy in epidemiological investigations, including T2DM risk reduction. Quercetin is a natural inhibitor or regulator of iron metabolism and is beneficial in improving diseases caused by iron overload. Studies have shown that quercetin treatment significantly restores GSH content and SOD activity in pancreas β cells. The results indicate that quercetin has a potential beneficial effect on T2DM, and functions by inhibiting pancreas β cells ferroptosis, highlighting the promising curative effect of quercetin in T2DM.

21Many studies have shown that melatonin is a potent endogenous antioxidant, which also indirectly stimulates certain antioxidant enzymes such as SOD and Gpx4. Recent studies have shown that melatonin reduces diabetic kidney injury and exerts neuroprotective effects by activating the Nrf2/HO-1 pathway and increasing the levels of antioxidant enzymes HO-1 and NAD(P)H dehydrogenase [quinone] 1 (NQO1). Epidemiological studies have shown that the incidence of osteoporotic fractures increases in T2DM patients compared with healthy populations. High glucose induces ferroptosis via increased ROS/lipid peroxidation/glutathione depletion in type 2 diabetic osteoporosis. Melatonin significantly reduced ferroptosis and improved the osteogenic capacity of MC3T3-E1 cells by activating the Nrf2/HO-1 pathway in vivo and in vitro.

22Studies have shown that iron overload in the pancreas contributes to T2DM pathogeneses. Vitamin D can inhibit ferroptosis in diabetic pancreatic β cells through NF-κB-DMT1 signaling, which is a potential protective drug in the development of T2DM.

23Systemic inflammation is mainly caused by activation of the myeloid differentiation factor 2 (MD2)/toll-like receptor 4 complex, a key mediator of left ventricular dysfunction in prediabetes. Study was shown that in obese mice the MD2 inhibitor L6H21 effectively reduced systemic inflammation, and L6H21 provides cardioprotective efficacy in a dose-dependent manner, by reducing apoptosis and ferroptosis. Furthermore, Ze450 has been shown to be protective against cellular peroxidation, where Ze450 retains mitochondrial function and integrity by inhibiting ferroptosis. Thus, promoting the ability of cells to recover from oxidative stress both in vitro and in vivo is a therapeutic opportunity for metabolic diseases such as T2DM. A novel molecule, M1, was found to enhance the lability of the Fe-2S clusters of mNT and NAF-1 proteins, reduced mitochondrial iron and ROS accumulation, and successfully treated diabetic mice.

24A growing number of evidence supports the role of ferroptosis in the initiation and progression of various metabolic diseases such as T2DM. However, several questions need to be addressed before the therapeutic potential of ferroptosis-targeted agents can be clinically evaluated. What are the crucial safeguarding mechanisms for ferroptosis in diabetes? Which are the reliable biomarkers for predicting ferroptosis in metabolic disease? Plasma ferritin concentration is currently used as a ferroptosis biomarker in preclinical studies. However, it is non-specific and is present in other types of cell death and several pathological conditions. The lack of ferroptosis-specific biomarkers has been a long-standing bottleneck, limiting the development of ferroptosis-targeted clinical applications. Finally, how does the interplay between ferroptosis and other forms of cell death affect the development of metabolic diseases? To date, no clinical trials have investigated ferroptosis-specific inhibitors for metabolic disease treatment. Most of the research uses selective inhibition of ferroptosis, which has been shown to substantially improve pancreatic β cells function in various animal models. Large population-based datasets are urgently needed to determine whether selectively blocking ferroptosis can improve T2DM and its complications.

25The relationship between iron metabolism and glucose homeostasis is now widely recognized. Iron has been shown to affect glucose homeostasis in organs and cells, such as pancreas β cells, hepatocytes, and adipose tissue. In addition, iron metabolism is related to the brain-gut axis and circadian rhythm. Iron metabolism disorders result in insufficient insulin secretion and IR; however, the relationship between iron metabolism and T2DM and its complications was unclear until the discovery of ferroptosis. Excess levels of free reactive iron cause tissue damage and oxidative cell death. Different drugs and compounds for lipid peroxidation have been widely studied for the treatment of T2DM induced by iron overload. However, there is a lack of clinical research at this stage and preclinical research is paving the way for the development of effective ferroptosis-specific antagonists for the clinical treatment of T2DM.