Although deranged iron metabolism has repeatedly been reported in association with NAFLD/MAFLD (reviewed in [38]), a cause-and-effect association remains unproven in observational studies. Wang et al. [38] conducted Mendelian randomization analyses to address this research question regarding causality. By utilizing data from the UK Biobank, the Estonian Biobank, the eMERGE network, and FinnGen consortium, these authors were able to conclude that, at sex-stratified analysis iron status predicted genetically was specifically associated with increased odds of NAFLD and FIB/cirrhosis in men [38].

In humans, iron fortification carries dysbiosis via an unfavorable ratio of fecal Enterobacteria to Bifidobacteria and Lactobacilli [39, 40]. Conversely, phlebotomy conducted among individuals with haemochromatosis is associated with a reduction of fecal iron and increased concentrations of bacterium Faecalibacterium prausnitzii which, in turn, is associated with improved colonic health [41].

Ten years ago, Dongiovanni and colleagues [42] shaped an experimental model of DIOS. These authors showed that, in C57Bl/6 male mice, a diet enriched in iron induces IR and hyperTG and affects the metabolism of visceral adipose tissue through up-regulation of hepcidin [42]. Another study conducted in vitro showed that free iron-induced an inflammatory response selectively in pre-adipocytes, documented by increased interleukin-6 secretion, which was blocked by co-incubation with either iron chelators or radical scavengers [43].

It is widely accepted that both dietary iron and iron overload are strong risk factors for diabetes and that this association is mechanistically mediated by pancreatic beta cell failure, IR, and regulation of energy metabolism in iron-sensing adipocytes [47]. While the molecular mechanisms mediating these effects are not fully understood, oxidant stress, modulation of adipokines, and intracellular signal transduction pathways are all deemed to play a role [47].

As regards the specific NAFLD arena, in 2002, Lonardo et al. [48] reported in their case-control study that—at univariate analysis the relative risk of NAFLD (defined as ultrasonographic evidence of steatosis in individuals without any competing causes of steatogenic liver disease) significantly decreased with increasing transferrin. However, at multiple logistic regression analysis, only fasting insulin and uric acid were found to be independent predictors of NAFLD [48]. The primacy of hyperinsulinemia in NAFLD has more recently been confirmed by Bril et al. [49] who have shown that fasting intact insulin concentrations (measured with mass spectrometry) accurately predict NAFLD among non-diabetic individuals.

In 2014, Wlazlo et al. [50] prospectively investigated the associations of biomarkers of iron metabolism with IR at various anatomic sites (e.g., muscle, liver, and adipocytes) and, importantly, with impaired glucose tolerance. To this end, a cohort of 509 individuals was followed up for a 7-year period. The study concluded that biomarkers of iron metabolism are associated with IR in muscle, liver, and adipocytes, eventually leading to hyperglycemia [50].

In 2020, Mayneris-Perxachs et al. [51] investigated the plasma lipidome, IR, biomarkers of iron metabolism, together with a profile of cytokines and adipokines in 65 overweight/obese individuals. Data have shown that several lipid classes belonging to the phosphatidylcholines, lisophosphatidylinositol, dihydroceramides, phosphatidylserine, diacyliglerols, and triacylglycerols lipid classes, were positively associated with serum ferritin concentrations, an established biomarker indicating iron storage. Given that many of these lipid classes are reportedly involved in IR and type 2 diabetes in both experimental studies and in humans, it appears logical to conclude that the entity of iron depots may modulate glucose metabolism through lipidomic changes. In this regard, the key issue is whether lipids or iron are major determinants of IR. To address this research question, Martin-Rodriguez et al. [52] conducted a cross-sectional study of 104 healthy non-diabetic Caucasians submitted to quantitative magnetic resonance imaging (MRI) T2 gradient-echo technique to gauge hepatic iron and ¹H-magnetic resonance (¹H-MR) spectroscopy to measure intra-hepatic triglyceride (TG; IHT) content. Data have shown that IR was more closely associated with IHT than intra-hepatic iron (although some interplay occurred between them). Interestingly, the association of NAFLD with IR was mediated by high serum TNF-α concentrations, while increased IHT appear to affect the development of hepatic iron overload in individuals who were free of both diabetes and hemochromatosis [52].

Gut microbiota plays a key role in metaflammation. In rats, a high-fat high-fructose (HFHF) diet induces hepatic metaflammation and metabolic disorders via disturbed homeostasis of gut microbiota and altered innate immune system, activation of lipopolisaccaride-Toll-like receptor 4 (LPS-TLR4) signaling pathway, and messengers such as α-hydroxyisobutyric acid and other organic acids [53]. Given its critical role in the development of metaflammation and metabolic disorders, it is important to investigate the relation of gut microbiota with iron metabolism. In this connection, whenever more iron is available in the gastrointestinal tract, the composition of intestinal microbiota is altered, with a reduced number of the beneficial Firmicutes species to the advantage of noxious species such as Proteobacteria, particularly Enterobacteriaceae [54].

Iron participates in vital biological pathways such as the red blood cell-mediated transport of oxygen and oxygen reduction to water during respiration. While this metal generally has a limited availability, pathological accumulation of iron within tissues elicits toxic effects via increased generation of ROS and enhanced oxidative stress [55]. Recent studies suggest that ferroptosis, which may be triggered by various stimuli notably including intracellular accumulation of iron and lipid peroxidation, underlies ROS-dependent regulated cell death [56]. Iron present in the cytosol promotes ROS formation through the Fenton reaction (i.e., H₂O₂ is transformed in hydroxyl radicals) which caused lipid peroxidation to play a key role in ferroptosis [56]. Additionally, lipid peroxidation occurring within the liver results in damage to hepatocyte mitochondria and lysosomes, which are deemed to participate in the processes of cell necrosis and apoptosis, ultimately leading to the fibrogenic process in the liver [57], which is also discussed below under point 4.8.

The ER defines the sub-cellular organelle that, through ER-resident enzymatic activities and chaperones, governs proper folding of linear polypeptides and proteins [58]. This is a vital process given that failure to shape the proper three-dimensional architecture of proteins will eventually lead to storage of misfolded or unfolded proteins. Perturbed ER homeostasis, eventually culminating in the so-called ER stress, is triggered either by accumulated misfolded/unfolded proteins or lipotoxicity. In response to ER stress, the unfolded protein response (UPR) is activated to reestablish ER homeostasis (hence considered to be an “adaptive UPR”), or, conversely, to culminate in cell death (in this case ER stress is “maladaptive”) [58]. UPR participates in a variety of pathophysiological states, spanning from inflammatory response, nutrient disorders, and viral infection as well as development and progression of various liver conditions, notably including NAFLD and others [58, 59].

Recent studies have highlighted the connections linking the liver hormone hepcidin with ER stress. High hepcidin concentrations (which may mirror iron body status, inflammatory signals, or ER stress) result in iron being stored intracellularly at various sites (e.g., duodenal enterocytes, hepatocytes, and macrophages) [59]. In other words, accumulation of liver fat induces ER stress which, in turn, promotes hepatic lipogenesis, thus creating a positive-feedback loop, which may not only contribute to the development of HS but also to the maintenance of hepatocellular injury and FIB progression within the pathogenic NASH spectrum [59].

Liver FIB describes the accumulation of extracellular matrix components that result from persistent liver injury. While the fibrotic hepatic response was selected by evolution to keep tissue integrity, whenever exceeding a physiological amount, it paradoxically impairs liver regeneration thus threatening hepatic health and function [60]. Irrespective of the type of inciting insult, repeated bouts of hepatocyte damage, followed by the wound-healing fibrosing response, may potentially result in progressive hepatic FIB disease as well as cirrhosis, namely a distorted liver histology exhibiting abundant amounts of scar tissue; development of nodular (as opposed to lobular) hepatic architecture and eventually resulting in the ominous triad of portal hypertension, liver failure, and primary liver cancer [60].

The clinical and mechanistic aspects of iron metabolism relevant to the development of hepatic FIB have been reviewed elsewhere [61]. Data indicate that excess iron deposition, which is frequently observed in chronic liver diseases owing to variable etiology, feeds the Fenton reaction to generate large amounts of free radicals that severely harm cells and tissues, ultimately perpetuating fibrosing signals both in the parenchymal and non-parenchymal liver cells [61]. Collectively, these phenomena exacerbate disease progression and aggravate liver histology.