Diabetes is a metabolic disorder characterized by hyperglycemia resulting from an absolute deficiency of insulin secretion (type 1 diabetes, T1D), or defective insulin secretion and/or insulin action (type 2 diabetes, T2D) [1]. In recent decades, T2D has become a global pandemic and a major healthcare burden worldwide [2]. Lifestyle and genetic factors leading to heme accumulation may contribute to the pathogenesis of T2D [[3], [4], [5], [6], [7]]. Although the exact mechanism for heme-mediated pathophysiological processes contributing to T2D is unclear, the evidence based on animal models suggests that ferroptosis is associated with insulin secretion dysfunction in pancreatic β cells [8]. Even so, the underlying mechanism of how heme impairs glucose-stimulated insulin secretion (GSIS) has not been conclusively established.

Tyrosine nitration, defined as the addition of a nitro group (-NO2) onto a 3-position carbon of the phenolic ring of free or protein-bound tyrosine to form 3-nitrotyrosine (3-NT), is an oxidative post-translational modification [9]. Tyrosine nitration has served as a biomarker of oxidative stress and is present in high abundance in over 50 disease pathologies in humans, including T2D [10]. Several in vivo studies have suggested that nitration of tyrosine in insulin receptor, insulin receptor substrate and Akt in skeletal muscle and liver may lead to impaired insulin signal transduction [11]. In addition to the effect on insulin signaling, tyrosine nitration also leads to β-cell dysfunction [12]. Lupi et al. [13] studied isolated human pancreatic islets from type 2 diabetic and non-diabetic subjects, matched for age and body mass index. They found that compared with non-diabetic islets, the concentration of 3-NT in diabetic islets increased, while GSIS decreased. This led us to hypothesize that the reduced insulin secretion is due to tyrosine nitration of specific proteins in the pancreatic islets. This is consistent with previous report that tyrosine nitration of specific target proteins can impair the stimulation of insulin secretion in β-cells [14].

Heme or heme peroxidases-dependent protein tyrosine nitration plays an important role in inflammatory sites [11,15]. In this tyrosine nitration mechanism, heme is oxidized by H2O2 to form compound I, a high oxidation state oxo-heme complex that oxidizes adjacent (either free or in protein) tyrosine to Tyr radical (•Tyr) and NO2− to NO2, generating the proper combination of reagents to yield 3-NT [[16], [17], [18]]. In addition, heme can interact with proteins through His, Tyr, Met or Cys to form heme-protein complexes [19,20]. This transient binding results in an impact on the function of proteins. Broadly two types of functional outcomes can be identified – gain of function and loss of function [21]. Although the potential role of heme is unclear, it can be speculated that in an inflammatory environment, heme-bound proteins are more prone to tyrosine nitration modification through heme-dependent catalytic mechanisms, affecting their biological activity.

Glucagon (GCG), a 29 amino acid peptide produced by the α cells in the pancreas, has received the most attention because of important roles in glucose metabolism [22]. The prominent role of circulating GCG to promote hepatic glycogenolysis and gluconeogenesis and raise blood glucose [22,23] has obscured paracrine actions of GCG on β cells. GCG is known to be stimulators of insulin release in β cell lines and pancreatic islets [[23], [24], [25], [26]]. Although each type of diabetes in animals and humans is accompanied by hyperglucagonemia [27], the new studies raise the possibility that the hyperglucagonemia present in T2D is a compensatory mechanism to enhance β-cell function, rather than induce dysregulated glucose homeostasis [[23], [24], [25]]. GCG-dependent insulin secretion is more apparent at high glucose levels, indicating that intra-islet GCG is particularly required at times with high insulin secretion demand, and further emphasizes the role of paracrine intra-islet GCG in maintaining appropriate insulin secretion [26,28,29]. However, in states of poorly controlled diabetes with severe insulin deficiency or ketoacidosis, plasma GCG concentrations are extremely high [30], indicating that paracrine intra-islet GCG actions to maintain appropriate insulin secretion may be impaired [31]. Although the reasons why the insulinotropic effect of GCG is impaired in T2D are not completely clear, studies on islets of T2D patients showed that compared with non-diabetic islets, the concentration of 3-NT in diabetic islets increased and insulin secretion stimulated by glucose decreased [13]. In addition, GCG contains up to four heme coordination residues (1His, 10Tyr, 13Tyr and 27Met) and five amino-acid residues (9Asp, 15Asp, 21Asp, 17Arg and 18Arg) that may increase heme peroxidase-like activity, as the presence of these amino-acid residues in the proximal or distal heme environments may significantly enhance the peroxidase-like catalytic activity of heme [32]. In addition, in inflammatory diseases including T2D, protein tyrosine nitration can occur by the action of enzyme catalysts, such as heme-containing peroxidases in the presence of NO2− and H2O2, which is considered one of the main pathways for protein modification in biological systems [11,33]. Herein, the aim of the present work is to extend these preliminary results by showing the nature of heme binding to GCG, catalyzing its oxidative posttranslational modification, and linking it with the decreased insulinotropic effect of GCG under diabetic conditions. Further, these results may expand our understanding of heme as a multifaceted effector molecule in the development of diabetes.