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The peptide hormone insulin is a key regulator of energy metabolism, proliferation and survival. Binding of insulin to its receptor activates the PI3K/AKT signalling pathway, which mediates fundamental cellular responses. Oxidants, in particular H2O2, have been recognised as insulin-mimetics. Treatment of cells with insulin leads to increased intracellular H2O2 levels affecting the activity of downstream signalling components, thereby amplifying insulin-mediated signal transduction. Specific molecular targets of insulin-stimulated H2O2 include phosphatases and kinases, whose activity can be altered via redox modifications of critical cysteine residues. Over the past decades, several of these redox-sensitive cysteines have been identified and their impact on insulin signalling evaluated. The aim of this review is to summarise the current knowledge on the redox regulation of the insulin signalling pathway.
Insulin, an anabolic peptide hormone secreted by pancreatic β-cells, is a key regulator of important metabolic processes such as glucose and lipid homeostasis, as well as a determinant of longevity. The actions of insulin are mediated by the so-called insulin signalling pathway, initiated by the binding of insulin to its receptor, which triggers a sequence of intracellular phosphorylation events . The insulin pathway maintains metabolic homeostasis by redirecting post-prandial glucose into muscle and adipose tissues, and by suppressing glucose production in the liver. Furthermore, insulin regulates the generation of energy storage, such as glycogen and triacylglycerides . In this context, insulin resistance is defined as the decreased ability of a tissue to adequately respond to the actions of insulin, which is a risk factor for developing type-2 diabetes (T2D). The worldwide prevalence of T2D has grown concerningly over recent decades, and is estimated to increase further from 9.3% in 2019 (463 million people) to 10.9% by 2045 (700 million people). Furthermore, T2D is an important risk factor for the development of co-morbidities including cardiovascular and kidney diseases, resulting in reduced quality of life . An undeniable connection exists in Western societies between behaviour, diet, obesity and T2D. The growing incidence of T2D is linked, at least partly, to decreased energy expenditure due to our increasingly sedentary lifestyles, and to increased consumption of processed foods and drinks containing high levels of sugar. The pathological hallmarks of T2D in mammals include: i) the resistance of peripheral tissues to insulin signals, leading to ii) hyperglycemia and compensatory hyperinsulinemia, and iii) impaired/abnormal insulin secretion by pancreatic β-cells . Most individuals with T2D are obese, and obesity itself can cause insulin resistance
Several studies have implicated the involvement of oxidative stress in the development of insulin resistance and T2D. Oxidative stress arises from the aberrant production or defective scavenging of reactive oxygen species (ROS), leading to damage of macromolecules . For example, ROS associated with several abnormalities, such as hyperglycemia, non-enzymatic glycosylation, inflammation and/or dyslipidemia, may cause decreased insulin expression and/or an impaired response to the insulin signal . Thus, the increased supply of energy substrates and the inflammatory environment under T2D conditions are thought to result in the excessive generation of mitochondria-derived ROS that suppress the insulin signalling cascade and thereby promote the development of insulin resistance.
Besides these harmful and cell damaging high levels of ROS, lower doses of ROS, in particular hydrogen peroxide (H2O2), play essential roles in fine-tuning the insulin signalling pathway and are therefore indespensible for its optimal functioning. Several phosphatases, including those counteracting the insulin-stimulated phosphorylation cascade, contain critical cysteine residues at their active site that can be oxidised in response to H2O2, resulting in their inactivation. Similarly, several kinases that mediate insulin signalling are prone to redox regulation.
n this review, we summarise the current state of knowledge on redox signalling events that contribute to regulating the insulin signalling pathway. Furthermore, we show that key redox-active cysteine residues are evolutionarily conserved from invertebrates to humans, which is consistent with their significant physiological role.
ROS is a commonly used term that includes both short-lived and more stable products from the reduction of molecular oxygen (O2). The step-wise transfer of single electrons during O2 reduction results in the formation of the superoxide radical anion (O2•–), as well as non-radical species such as H2O2 [15]. In the presence of iron ions, H2O2 can form the hydroxyl radical (•OH), which is highly reactive (diffusion-limited) and therefore more implicated in oxidative damage rather than redox signalling. Superoxide is generated ‘accidently’ by the mitochondrial respiratory chain as a by-product of aerobic metabolism. Moreover, superoxide can be produced deliberately by the NADPH oxidase (NOX) family of enzymes in response to stimuli, including growth factors such as insulin . H2O2 is the most abundant ROS in eukaryotes with a cellular steady-state concentration of ~1–10 nM. H2O2 is formed from superoxide by the action of superoxide dismutase (SOD) enzymes . H2O2 can also be produced directly, for instance by xanthine oxidase in purine catabolism or by the endoplasmic reticulum oxidoreduction ERO1 during disulphide bond formation by the protein folding system.
While excessive formation of ROS is associated with the development of many diseases in humans including T2D, it has become increasingly apparent over recent years that low levels of specific ROS, particularly H2O2, are required for normal cellular function, and are involved in the regulation of many physiological processes such as signal transduction, cell differentiation and proliferation. H2O2 is formed enzymatically by NOXs together with SODs in response to growth factor signals, e.g. insulin. This localised transient H2O2 burst is essential for optimal tyrosine-phosphorylation-dependent signalling events by modifying the activity of kinases. These include the insulin receptor (IR) kinase domain itself, as well as phosphoinositide 3-kinase (PI3K) and AKT. Furthermore, the activity of phosphatases, such as PTEN, PTP1B and PP2A, that counteract insulin signalling by dephosphorylating and thereby inhibiting insulin-responsive kinases, is modulated by H2O2 (see Section 4.3). Thus, the localisation and levels of ROS have an impact on the progression of the insulin signalling cascade.
Insulin plays an essential role in controlling nutrient and metabolic homeostasis. Insulin promotes glucose storage in the liver and glucose uptake into fat and muscle cells via the glucose transporter GLUT4. In response to insulin, GLUT4 is translocated from the cytoplasm to the plasma membrane. Mice lacking GLUT4 are insulin resistant . The precise mechanism whereby insulin leads to the translocation of GLUT4 is still not fully understood, but the PI3K/AKT pathway is known to play an essential role.
The insulin receptor (IR) is a α2β2 heterotetrameric glycoprotein belonging to the receptor tyrosine kinase superfamily, with 20 sub-families described in humans, based on sequence homology, structure and ligand affinity. Besides the IR, other members of the receptor tyrosine kinase superfamily include endothelial growth factor receptor (EGFR) with its ligand EGF, and platelet-derived growth factor receptor (PDGFR) with its ligand PDGF .
Chronically high ROS associated with hyperglycemia are recognised to have pathophysiological roles in the progression of T2D, e.g. impairment of β-cell function, or the development of further co-morbidities such as vascular complications. Studies in healthy humans and rodents further revealed that the adipose tissue experiences oxidative stress as a result of excessive caloric intake leading to the development of insulin resistance. Using a model of physiologically derived oxidative stress by inhibiting TrxR and GR simultaneously in adipocytes, >2000 genes were found to be differentially expressed compared to untreated cells. Interestingly, this response shared many similarities with changes observed in insulin resistance models. Providing these cells with an antioxidant induced only minor transcriptional changes, but rescued the insulin resistance. This indicates that the transcriptional changes observed in response to oxidative stress are not the cause of insulin resistance. Thus, oxidative stress must have effects in addition to transcriptional changes to cause insulin resistance.
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Claudia Lennicke a b, Helena M. Cochemé
James Eckburg
