Impairment of the IR-IRS-PI3K-AKT signaling pathway in Insulin Resistance: New Insights into Insulin Resistance Mechanisms

Impairment of the IR-IRS-PI3K-AKT signaling pathway in Insulin Resistance: New Insights into Insulin Resistance Mechanisms | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 12 September 2025 V1 Latest version Share on Impairment of the IR-IRS-PI3K-AKT signaling pathway in Insulin Resistance: New Insights into Insulin Resistance Mechanisms Author : Syed Sohail Ahmad 0009-0005-5129-3864 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175767205.55981786/v1 1861 views 213 downloads Contents Abstract Abstract Introduction IRS Signaling Dysfunction PI3-K Signaling Pathway 1.3 PI3-kinase regulatory subunits PDK1 and AKT/PKB pathway Peroxisome proliferator–activated receptor gamma (PPAR-γ) Tumor necrosis factor-alpha TNF–α Discussion TNF-α Neutralization PPAR-γ(P115Q) mutation Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Insulin is a master metabolic hormone that regulates the metabolism of glucose and fat. Obesity produces factors that increase insulin resistance, which is the leading cause of type 2 diabetes in the world. In insulin resistance, the main signaling pathway IR-IRS-PI3K-AKT pathway, is disrupted, which is the central cause of insulin resistance and T2DM. This review explains the molecular mechanism of the dysfunction of these signaling pathways. Focusing on receptor dysfunction, post-receptor alteration, and all other factors that decrease signaling accuracy. The importance of genetic modifiers and receptor–substrate dynamics is shown by the fact that combined IR and IRS-1 knockouts mimic human type 2 diabetes. IRS-1 serine phosphorylation disrupts IR–PI3K interaction, which reduces downstream signaling. We study different AKT isoforms. There is a role of AKT-2 in insulin resistance, but a little role of AKT-1and AKT-3. Furthermore, we study a rare form of mutation, PPAR-ℽ mutation (P115Q), which shows that despite severe obesity, there is insulin sensitivity and lower insulin resistance. TNF-α signaling inhibition, which raises receptor tyrosine kinase activity in muscle and fat, restores insulin sensitivity. This review shows that insulin resistance is not caused by a single factor, but rather by a combination of different proteins which is modified and unable to function properly, such as loss of Tyrosine Kinase activity, nuclear receptor modulation, IRS dysregulation, receptor dysfunction, PI3-K dysfunction, AKT protein disturbance, and inflammatory stress. Understanding these dysfunctions, various unknown factors, and processes enables the development of precision therapies that can restore metabolic balance and slow the progression of type 2 diabetes. Title Impairment of the IR-IRS-PI3K-AKT signaling pathway in Insulin Resistance: New Insights into Insulin Resistance Mechanisms Author Affiliation: Corresponding Author Contact: Abstract Insulin is a master metabolic hormone that regulates the metabolism of glucose and fat. Obesity produces factors that increase insulin resistance, which is the leading cause of type 2 diabetes in the world. In insulin resistance, the main signaling pathway IR-IRS-PI3K-AKT pathway, is disrupted, which is the central cause of insulin resistance and T2DM. This review explains the molecular mechanism of the dysfunction of these signaling pathways. Focusing on receptor dysfunction, post-receptor alteration, and all other factors that decrease signaling accuracy. The importance of genetic modifiers and receptor–substrate dynamics is shown by the fact that combined IR and IRS-1 knockouts mimic human type 2 diabetes. IRS-1 serine phosphorylation disrupts IR–PI3K interaction, which reduces downstream signaling. We study different AKT isoforms. There is a role of AKT-2 in insulin resistance, but a little role of AKT-1and AKT-3. Furthermore, we study a rare form of mutation, PPAR-ℽ mutation (P115Q), which shows that despite severe obesity, there is insulin sensitivity and lower insulin resistance. TNF-α signaling inhibition, which raises receptor tyrosine kinase activity in muscle and fat, restores insulin sensitivity. This review shows that insulin resistance is not caused by a single factor, but rather by a combination of different proteins which is modified and unable to function properly, such as loss of Tyrosine Kinase activity, nuclear receptor modulation, IRS dysregulation, receptor dysfunction, PI3-K dysfunction, AKT protein disturbance, and inflammatory stress. Understanding these dysfunctions, various unknown factors, and processes enables the development of precision therapies that can restore metabolic balance and slow the progression of type 2 diabetes. Key words Insulin resistance, Insulin receptor signaling, IRS phosphorylation, Akt isoforms TNF-alpha and PPAR-gamma modulation, Type 2 diabetes, Metabolic dysfunction, Targeted therapies Introduction When the body’s tissues no longer react to insulin, normal insulin levels are unable to produce their usual biological effects, leading to the development of insulin resistance. This response is frequently observed in conditions such as obesity and type 2 Diabetes.[1] Some of the biggest obstacles to biochemical research are identifying the critical processes that result in insulin signaling specificity. Nevertheless, the results should provide novel therapeutic strategies for the management of individuals with insulin-resistant conditions, such as type 2 diabetes [2]. Blood glucose levels in these patients are lowered by intensive insulin therapy. Nonetheless, these patients have many complications, including heart disease, neuropathy, retinopathy, microvascular and macrovascular problems, and more, as a result of their elevated body weight and insulin resistance. Understanding the action of insulin signaling mechanisms and finding an effective management strategy for metabolic syndrome, type 2 diabetes mellitus, and associated cardiovascular dysfunction has important clinical implications. The biological effects of insulin are caused by a transmembrane tyrosine kinase-mediated intracellular signaling pathway that includes the insulin receptor. When insulin binds to the alpha-subunit, the receptor dimerizes to form the α2β2 complex in the cell membrane. The first step in activating IR is the autophosphorylation of the β-subunit at Tyr1158, Try1162, and Tyr1163. PI3K activity linked to IRS-1. Conversely, in the skeletal muscle of individuals with type 2 diabetes who are obese, IRS. When IR tyrosine kinase is activated, it recruits and phosphorylates several substrates, such as IRS1-4, SHC, Grb-2-associated protein (GAB1), DOCK1, CBL, and APS adaptor proteins. These substrates all offer particular docking sites for the recruitment of other downstream signaling proteins, which in turn activate the phosphatidylinositide-3-kinase (PI3K)/Akt signaling cascade and Ras/MAPKs.[3] Multi-subunit signaling complexes are created when the cytosolic proteins insulin receptor substrate (IRS)-1 and IRS-2 undergo tyrosine phosphorylation, which creates protein scaffolding for the assembly of additional effector proteins [4]. Insulin receptor substrate (IRS)-1 travels to the cell membrane and gets phosphorylated on adjacent tyrosine molecules after the insulin receptor has been phosphorylated. The p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase is activated by tyrosine phosphorylation of IRS-1, which also activates the p110 catalytic subunit, increasing phosphatidylinositol-3,4,5 triphosphate. As a result, downstream protein kinase B, also known as Akt, is activated, and Akt substrate 160 (AS160) is phosphorylated, facilitating GLUT4’s translocation to the sarcolemma and the subsequent uptake of glucose into the cell as shown in Figure 1 [5]. Although PI3K activity linked to both IRSs is compromised, IRS-1 expression is decreased in adipocytes from obese people with type 2 diabetes, resulting in decreased -1 and IRS-2 normal protein levels [6]. Insulin Receptor IR Structure and Signaling Pathway A transmembrane unit, two intracellular beta-subunits, and two extracellular alpha-subunits constitute the complex tetrameric protein known as the insulin receptor (IR). It is a member of the receptor tyrosine kinase (RTK) subfamily, which also includes the insulin growth factor-1 receptor (IGF1-R) and the IR-related receptor (IRR), an orphan receptor [7]. The structure of the insulin receptor and the insulin-like growth factor-1 (IGF-1) receptor are similar, and they can even form chimeric hybrids. Physiologically. IGF-1 and insulin, however, have quite different purposes. It is still unknown how this happens, but one theory is that the insulin receptor has an extra phosphorylation site in its COOH-terminus that is absent from the IGF-1 receptor. This site might interact with PI3-kinase or other Sh2 proteins.[8] A unique gene creates each of these receptors. Two isoforms of the IR messenger RNA (mRNA) are generated by alternative splicing of exon 11; these are differentiated by the inclusion (isoform B) or exclusion (isoform A) of a 12-amino acid sequence in the carboxy-terminal portion of a subunit [9]. With a higher affinity for both insulin and IGF-2, a higher rate of internalization than the type-B isoform, and a tendency to be up-regulated in cancer, IR-A is mainly expressed in fetal tissues and the brain [10]. Furthermore, exon 11, which codes for 12 amino acids close to the C terminus of the α-subunit, is either absent (IR-A) or present (IR-B) in the two isoforms of the α-sub-unit that result from differential mRNA splicing. The expression patterns of the IR-A and IR-B isoforms differ, and the IR-B isoform is more likely to bind to IGF-1. Furthermore, IR-A and IR-B in pancreatic β-cells have been linked to different downstream events associated with insulin transcription and cell survival. [11] For instance, the kinase activity in the α-subunit is activated when insulin binds to the α-subunit or when the β-subunit is removed by proteolysis or genetic deletion. There is an orderly sequence in which the tyrosine clusters are phosphorylated. A phosphotyrosine-binding (PTB) domain and an NH2-terminal pleckstrin homology (PH) domain are features of IRS proteins. A COOH-terminal tail with several tyrosine and serine/threonine phosphorylation sites follows [12]. The phosphorylated NPXpY motif (Asn-Pro-Xaa-Tyr (pi); X is any amino acid and pi is inorganic phosphate) of the active IR is bound by the PTB domain, while the PH domain mediates connections between cell membranes. Each IRS protein has approximately 20 possible tyrosine phosphorylation sites at its COOH terminal, which serve as on/off switches to transduce insulin action. These sites attract downstream signaling proteins, such as phosphotyrosine phosphatase SHP2 and PI3K subunit, as well as adaptor molecules like GRB2, SOCS3, NCK, CRK, SH2B, and others [13]. The juxta membrane region is phosphorylated soon after the tyrosine in the kinase domain’s activation loop is phosphorylated. Tyrosine in the C-terminal domain is phosphorylated last [14]. The first step in activating IR is the autophosphorylation of the β-subunit at Tyr1158, Try1162, and Tyr1163, as well as the dimerization of the receptor to create the α2β2 complex in the cell membrane as a result of insulin binding to the α-subunit. The three tyrosines of the A-loop need to be phosphorylated for full activity, and there is evidence of a graded activation as each one is modified. The monophosphate form is almost inactive, the bisphosphate form is partially active, and the trisphosphate-kinase is fully active. [15] Following this initial activation, there is transphosphorylation of the β-subunits, which leads to a conformational change and further increase in kinase activity. Phosphotyrosine at site 960 of the β-subunit just inside the membrane creates an NPXpY-recognition motif for the PTB domain of the IRS proteins [16]. In mice, disruption of the gene for IRS-3 alone does not result in abnormalities, but leads to a severe defect in adipogenesis when combined with deletion of IRS-1 [17]. IRS-4 mRNA is present in skeletal muscle, liver, heart, brain, and kidney, and IRS-4 KO mice show only very minimal growth retardation and glucose intolerance [18]. IRS-5 (also called DOK4) and IRS-6 (DOK5) have limited tissue expression and are relatively poor IR substrates [19]. During interaction with the insulin receptor, the IRS proteins are phosphorylated on several tyrosine residues by the insulin receptor, creating binding sites for multiple SH2 domain proteins, as shown in Figure 1 [16]. Furthermore, there are several kinds of negative regulation systems. Tyrosine phosphatases are one family of regulatory proteins; PTP1B, for example, is the most well-studied of them. PTP1B reduces the IR’s activity by directly interacting with it and dephosphorylating important residues of tyrosine. Through enhanced insulin signaling, the PTP1-B knockouts showed remarkable effectiveness in raising insulin sensitivity in vivo. By sterically hindering its interaction with the IRS proteins or modifying its kinase activity, other proteins, such as plasma-cell-membrane glycoprotein-1 (PC-1), growth-factor-receptor-bound protein 10 (Grb-10), and suppressor of cytokines signaling -1 (SOCS-1) and SOCS-3, downregulate IR function. Since the SOCS proteins are uncontrolled in insulin-resistant conditions like obesity, they may have a role in the pathogenesis of diabetes, making them especially significant. A common characteristic of the majority of insulin-resistant, hyperinsulinemic conditions, such as obesity and type 2 diabetes, is the downregulation of the IR at the protein level due to ligand-stimulated internalization and degradation. Insulin Receptor Substrates (IRS) Signaling Pathway The IRS is a key location for both positive and negative regulation of insulin signaling transduction and is an essential modulator of insulin action. From IRS-1 to IRS-6, the six members of IRS have comparatively similar gene sequences and three-dimensional structures. Because its receptor association is temporary, many IRS proteins can bind to a single active IR sequentially, each of which will become tyrosine phosphorylated during the relationship before forming a separate signaling complex. As a result, the receptor can enhance its signal by using IRS proteins. More importantly, the signal can be physically transferred to different areas of the cell by active IRS proteins, which carry their cargo of activated PI-3-kinase with them.[20] For instance, insulin stimulates the translocation of activated IRS-1/PI-3-kinase complexes to internal vesicles, which are abundant in glucose transporters and originate from the tubulovesicular endosomal membrane system [21]. However, IRS-1 (activated by IGF-IR) translocates to the nucleus upon IGF-I stimulation; this migration is crucially dependent on IRS-1’s PTB domain [22]. Furthermore, the IR and IRS proteins are regulated similarly: tyrosine phosphorylation activates them, and protein tyrosine phosphatases (PTPs), serine phosphorylation, and ligand-induced down-regulation negatively regulate them [23]. The C-terminal domain, the adjacent phosphotyrosine-binding (PTB) domain, and the pleckstrin-homology (PH) domain are all very similar. The NPXpY sequence of IR is bound by the PTB domains, and the C-terminal domain contains about 20 possible tyrosine phosphorylation sites. These sites can become phosphorylated upon IR activation and attach to proteins that include the Src homology domain 2 (SH2), including the tyrosine protein phosphorylase SHP-2, Grb-2 protein, and the p85α subunit of the PI3K protein. All proteins in this family, except the Shc, include the NH2-terminal PH domain, which aids in the targeting of the IRS proteins to the membrane and the insulin receptor. Similar to IRS-1, Shc binds to the active insulin receptor and undergoes phosphorylation. Even in the absence of a PH domain, phosphorylation is made feasible by the unique PTB domain found in Shc. Additionally, Shc only has one phosphorylation site that binds the adaptor protein Grb-2, in contrast to other members of the insulin receptor substrate family. This may result in the stimulation of the mitogenic signaling pathway and the activation of the Ras/MAP kinase (MAPK) pathway. Insulin is a weaker activator of Shc tyrosine phosphorylation than other growth factor stimuli [24]. Like the IRS proteins, Gab-1 is a high molecular weight protein without a PTB domain but with a PH domain and several phosphorylation sites. The insulin receptor phosphorylates Gab-1 modestly, while the epidermal growth factor receptor phosphorylates it extensively. Gab-1 is expressed in the tissues of various mammals, although its biological role is unknown. A new SH2 domain protein called Grb-IR has the ability to go from the cytosol to the plasma membrane and attach itself directly to the tyrosine-phosphorylated insulin receptor. Grb-IR uses its SH2 domain to attach to the crucial NPEpY960 in the insulin receptor’s juxtamembrane region. It has another binding site in the insulin receptor’s phosphorylated kinase activation loop, but the insulin receptor only very weakly phosphorylates it. Grb-IR overexpression suppresses PI3-kinase activation and the mitogenic effects of insulin and IGF-1. Whether it merely inhibits the insulin receptor or if its interaction with the insulin receptor has unique biological consequences is unknown [25]. Each isoform of IRS is a crucial node in insulin signaling pathways, and its absence causes a variety of physiological outcomes. While IRS2 gene knockout has no effect on cell differentiation but results in non-responsiveness to insulin-stimulated glucose transport, IRS1 gene knockout results in aberrant cell differentiation in preadipocytes. While IRS2 gene knockout animals exhibit insulin shortage primarily in the liver, causing growth problems in a few tissues, such as neurons and pancreatic cells, IRS1 gene knockout mice display insulin deficiency in muscle tissue. Studies on IRS1 and IRS2 tissue-specific knockouts in the liver have shown that while they both have complementary effects on activating the AKT signaling pathway, they have different functions in controlling gene expression. Hyperinsulinemia can reduce intracellular levels of the IRS1 and IRS2 genes in cell culture models and mouse tissues. The following is the specific mechanism of action: at the transcriptional level, hyperinsulinemia causes the degradation of IRS1 protein and inhibits the production of IRS2 [26]. Serine hyperphosphorylation of IRS1 is thought to be a negative regulator of insulin signal transduction generally, and serine phosphorylation of IRS1 rises with insulin resistance. Insulin resistance is exacerbated by elevated serine phosphorylation levels of IR and IRS, which are brought on by elevated circulating fatty acids and ectopic lipid accumulation in the liver and muscle [27]. Elevated levels of serine/threonine phosphorylation in IRS1 decrease IRS1 affinity for the p85 regulatory subunit of PI3K, decreasing insulin signal transduction and resulting in the symptom of insulin resistance [28]. Additionally, IRS1 phosphorylation at Ser636/639 and Ser307 prevents IRS1 from binding to IR. A negative charge is produced by serine/threonine phosphorylation at particular locations on the IRS, which modifies protein interactions and inhibits downstream cell signaling. Phosphorylation at specific locations in IRS causes conformational changes that increase downstream signaling and encourage protein interaction. Furthermore, the effects of phosphorylation at particular serine/threonine residues may vary based on the interaction status, the time course of insulin action, and the implications of temporal fluctuations in IRS phosphorylation. Ser-302/318 phosphorylation is linked to increased insulin signaling in the early phases of insulin action, but it is also required to reduce insulin activity in the later phases. Understanding insulin resistance will be aided by research on the regulatory effects of serine/threonine phosphorylation patterns on IRS1 activity [29]. Adaptor molecules, such as the regulatory subunit of PI3K or the adaptor molecule Grb2, which binds to son-of-sevenless (SOS) to activate the Ras–MAPK pathway, are the most well-studied SH2 proteins that bind to phosphorylated IRS proteins. When insulin is present, the insulin receptor (IR) phosphorylates insulin receptor substrate proteins (IRS-proteins), which are connected to the activation of two major signaling pathways: the Ras–Ras-Ras-mitogen-activated protein kinase (MAPK) pathway, which controls the expression of certain genes and works in tandem with the PI3K pathway to regulate cell growth and Figure 1. Schematic Diagram of IR-IRS-PI3K-AKT Signaling Pathway. Insulin binds with IR and activates signaling cascades. IRS binds with the SH2 domain, activates PI3k, which converts PIP2 to PIP3. Active PIP3 activates PDK1—activates AKT—which activates several pathways—AS60—GLUT-4 and absorbs glucose from the blood. IGF-1 activates the Ras/MAPK pathway, which helps in cell proliferation and growth. differentiation, and the phosphatidylinositol 3 kinase (PI3K) AKT/protein kinase B (PKB) pathway, which is in charge of the majority of insulin’s metabolic actions. IRS Signaling Dysfunction IRS proteins are phosphorylated by serine. Additionally, insulin and other stimuli, including cytokines and free fatty acids, cause IRS proteins to become serine phosphorylated. In general, serine phosphorylation appears to negatively inhibit IRS signaling, and there are more than 70 possible serine-phosphorylation sites in IRS1. In insulin-resistant conditions, serine phosphorylation of IRS1 is elevated and may contribute to the etiology of insulin resistance. IRS 1-knockout mice exhibit a widespread deficit in body growth due to IGF1 resistance and impaired insulin action, mainly in the muscle. IRS 2-knockout animals have aberrant growth in only a few organs, including certain neurons and pancreatic beta-cells, and have more severe abnormalities in insulin signaling in the liver. Similarly, IRS 2-knockout pre-adipocytes develop correctly but do not react to insulin-stimulated glucose transport, while IRS 1-knockout pre-adipocytes exhibit problems in differentiation at the cellular level. [30] It’s unclear exactly how serine phosphorylation modifies IRS1 function. One explanation could be that the phosphorylated serine disrupts the IRS1 functional domains where it is located. For example, it has been shown that negative control of insulin signaling is associated with the phosphorylation of Ser307 of IRS1, which is found in the PTB domain [31]. According to these knockout studies, the IRS proteins are a crucial node, meaning that each isoform’s deletion has a distinct biological impact. Since they are unable to activate MAPK and PI3K to the same extent as IRS1 and IRS2, IRS3 and IRS4 most likely alter the actions of IRS1 and IRS2, and they may even counteract parts of their functions when produced at high levels. Additionally, the cellular compartmentalization and activation kinetics of the IRS-protein isoforms vary. Furthermore, IRS2 differs structurally from the other IRS proteins; it has a distinct kinase-regulatory-loop binding domain that allows it to attach to the IR, which may help explain its particular function. Despite having normal glucose tolerance, IRS-1 knockout (KO) mice exhibit growth retardation and reduced insulin action, particularly in muscle [32]. IRS-2 KO mice have impaired insulin signaling in the liver, which, when paired with the loss of b cells, leads to the development of diabetes [33]. They also show growth reduction in specific organs, such as particular neurons and islet cells. While IRS-2 KO preadipocytes differentiate normally but have reduced insulin-stimulated glucose transport, IRS-1 KO preadipocytes have problems in differentiation at the cellular level [34]. Results from mice with specific gene ablations provide fascinating clues regarding the interactions between various IRS components and insulin and IGF-1 receptors. Findings from mice with targeted gene ablations offer intriguing hints about how different IRS molecules interact with insulin and IGF-1 receptors. Insulin or IGF-1 receptor ablation causes early postnatal death from diabetes or dwarfism with failure to thrive, respectively [35]. While ablation of IRS-2 results in death from a combination of insulin resistance and failure to develop a compensatory response of beta-cell levels, ablation of IRS-1 causes growth retardation and mild insulin resistance. This suggests that hyperglycemia is caused by the beta cell’s incapacity to compensate for peripheral insulin resistance. [36] Despite the substantial correlation between insulin resistance and serine phosphorylation of IRS1, its precise function in the pathogenesis of insulin resistance remains unclear. Furthermore, the potential regulatory significance of serine phosphorylation of other IRS proteins remains unclear.[26] First, elevated insulin levels cause the IRS1 protein to degrade and prevent the transcriptional synthesis of IRS2. Second, research has indicated that SOCS proteins may cause IRS1 and IRS2 to degrade by ubiquitylation. Regardless of the exact mechanism, in both humans and animals with diabetes, lower levels of IRS proteins and the IR itself undoubtedly contribute to insulin resistance. [3] PI3-K Signaling Pathway There are several isoforms of the regulatory and catalytic subunits that make up the PI3-K enzyme. Src homology 2 (SH2) domains are present in the majority of the intracellular partners of the insulin receptor substrates. These domains are similarly phosphotyrosine binding cassettes, but they may recognize particular amino acid sequences and have a higher binding affinity than PTB domains, allowing for a more rigid protein–protein connection. A highly conserved phosphotyrosine binding pocket (FLAVRES sequence) is one of the approximately 100 amino acids that make up SH2 domains [38]. A few amino acids COOH-terminal to the phosphotyrosine control the binding’s selectivity. Tyrosine-phosphorylated IRS-1 contains at least four pYMXM motifs that are recognized by the PI3-kinase SH2 domains. Other sequences, such as pYVNI, pYIDL, and pYASI sequences, are bound by the SH2 domains of the adaptor protein Grb-2 and the phosphotyrosine-sine- sine phosphatase SHP-2 (1). Together with these adaptor proteins, other SH2 proteins that bind to tyrosine residues on IRS proteins via their distinct SH2 domains include Crk (adaptor), Nck (adaptor), Fyn (tyrosine kinase), and Csk (tyrosine kinase). These SH2 adaptor proteins frequently contain SH3 domains. SH3 domains establish a connection between the adaptor protein and its downstream targets or related catalytic subunits by binding to proline-rich regions with the consensus sequence PXXP with a particular helix structure.[39] Other SH2 proteins, including Crk (adaptor), Nck (adaptor), Fyn (tyrosine kinase), and Csk (tyrosine kinase), bind to tyrosine residues on IRS proteins via their own SH2 domains in addition to these adaptor proteins. 1.3 PI3-kinase regulatory subunits A crucial component of the system that leads to insulin’s metabolic actions is PI3-kinase, a lipid kinase. The two components of PI3-kinase are a catalytic subunit that phosphorylates phosphatidylinositols present in cellular membranes and a regulatory subunit that binds to insulin receptor substrates. Tyrosine phosphorylated IRS proteins must bind to the two SH2 domains in the regulatory subunits for the PI3K to be recruited and activated. [40] As a result, the catalytic subunit is activated, phosphorylating phosphatidylinositol 4,5-bisphosphate (PIP2) quickly to produce phosphatidylinositol (3,4,5)-triphosphate (PIP3), the lipid second messenger, as shown in Figure 1. Akt is drawn to the plasma membrane by the latter, where it undergoes phosphorylation and triggers downstream signaling. Three separate genes encode the various isoforms of the PI3K regulatory subunit. All 65–75 of the regulatory subunits are encoded by Pik3r1, primarily as p85a but also as the splice variants p55a and p50a. Pik3r2 is responsible for 20 of the regulatory subunits and encodes p85b. P55g, which is encoded by Pik3r3, shares structural similarities with p55a but is expressed at low levels in the majority of tissues. Three distinct genes are responsible for the production of the three distinct catalytic subunits, p110a, p110b, and p110d. A catalytic subunit’s stability is increased, and it remains inhibited when a regulatory subunit binds to it. This is alleviated by the regulatory subunit’s activation upon binding to particular phosphotyrosine motifs in IRS proteins [41]. Particularly to the liver, Mice with p110a and, to a lesser extent, p110b ablation develop insulin resistance and glucose intolerance.[42] Thus, depending on its unique affinity for IRS proteins and capacity to control PI3-kinase activity, each regulatory subunit may have a distinct role. Additionally, each isoform can be involved in a particular subcellular compartmentalization [43]. Using a dominant-negative mutant or pharmaceuticals like wortmannin or LY294002 [44] to block PI3-kinase activity eliminates insulin-stimulated glucose uptake and prevents the translocation of GLUT4 vesicles to the plasma membrane. PI3-kinase suppression also inhibits many other cellular effects of insulin, including antilipolysis, fatty acid synthesis activation, acetyl-CoA carboxylase, glycogen synthase, Akt phosphorylation, glycogen synthase kinase 3 β-inactivation, and stimulation of protein and DNA synthesis. Numerous other hormonal cues that differ from the action of insulin can also activate PI3-kinase. This begs the question: What characteristics of PI3-kinase activation are unique to insulin? First, unlike other growth factors, the insulin receptor requires docking proteins, like IRS proteins, as a means of binding PI3-kinase instead of directly, as a relay mechanism. Unlike other growth factors, this causes PI3-kinase to be activated in a different cell compartment. In fact, it has been documented that after insulin stimulation, PI3-kinase activity moves from the cytosol to intracellular membranes. [45] Second, compared to the plasma membrane, the endoplasmic reticulum’s membrane surface area is larger, providing a larger pool of lipid substrates for PI3-kinase. Therefore, compared to other hormones, the insulin signal may produce more PI3-kinase products in vivo.[46] Csk . By tyrosine phosphorylation, the cytoplasmic tyrosine kinase known as COOH-terminal Src kinase (Csk) renders Src-type kinases inactive. According to reports, Csk binds to IRS-1 via its SH2 domain and, in an insulin-dependent manner, promotes the dephosphorylation of the focal adhesion kinase (FAK). One of the main participants in integrin and other growth factor signaling pathways is FAK, which facilitates interactions between cells and the extracellular matrix. Therefore, by modifying FAK activity in insulin signaling, Csk most likely contributes to the insulin-induced reorganization of cytoskeletal components [47]. PDK1 and AKT/PKB pathway A subset of the AGC protein kinase family, which includes isoforms of Akt/protein kinase B (PKB), p70 ribosomal S6 kinase (S6K), serum- and glucocorticoid-induced protein kinase (SGK), and multiple isoforms of protein kinase C (PKC), especially the atypical PKCs, mediate the majority of the physiological effects of PI3K-generated PIP3. Members of the AGC kinase family have similar structures and activation methods that involve phosphorylating two serine and threonine residues. [48] The primary upstream kinase in charge of phosphorylating and activating the AGC kinase members regulated by PI3K is PDK-1 (3-phosphoinositide-dependent protein kinase 1) [49]. When membrane-bound PIP3 binds to the PH domain of PDK-1, PDK-1 is activated as shown in Figure 1. AGC protein kinases are phosphorylated and activated by PDK-1 at serine/threonine sites, including Thr-308 for Akt [50]. However, complete activation necessitates Akt phosphorylation at Ser-473, which is achieved by the mammalian target of rapamycin complex 2 (mTORC2). [51] In response to DNA damage, DNA-dependent protein kinase (DNAPK) has also been shown to phosphorylate and activate Akt [52]. It also plays a role in insulin control of metabolic genes, such as fatty acid synthase. Every isoform has a PH domain, which enables recruitment to the plasma membrane and interaction with PIP3. Insulin-sensitive tissues have the highest levels of Akt2, which appears to be a key mediator of the effect of insulin on metabolism. In contrast to Akt1 and Akt3 KO mice, Akt2 KO mice exhibit insulin resistance and develop diabetes [53]. Many downstream targets can be phosphorylated and activated when PDK-1 and mTORC2 activate Akt. Tuberous sclerosis complex protein 2 (TSC-2) is phosphorylated by Akt, which causes the tumor suppressor complex made up of TSC-2 and TSC-1 to degrade. This activates the mTORC1 complex. Phosphorylation of proline-rich Akt substrate 40 KDa (PRAS40), an inhibitor of mTORC1, can also result in Akt-induced activation of mTORC1, removing the inhibition. A network of genes governing metabolism, protein synthesis, and cell growth is subsequently regulated by the mTORC1 complex, which also phosphorylates and inhibits 4E-binding protein 1 (4E-BP1) and activates the ribosomal protein S6 kinases S6K1 and S6K2, and SREBP1[54] Alterations in protein–protein interactions in insulin-resistant states. According to genomic studies, the pathogenesis of typical type 2 diabetes and obesity does not significantly include mutations of the insulin receptor itself [55]. IRS-1. The first insulin receptor substrate discovered to have several naturally occurring polymorphisms was IRS-1. Patients with type 2 diabetes are far more likely than controls to have polymorphisms of IRS-1, such as the G972R (glycine 972-arginine), S892G, G819R, R1221C, and A513P variations. The G972R polymorphism is the most prevalent and well-researched of these. The prevalence of this polymorphism is 5.8 in healthy individuals and 10.7 in patients with type 2 diabetes, respectively, and it is present in Caucasian populations. Obese carriers of this polymorphism exhibit reduced insulin sensitivity in oral glucose tolerance tests in Caucasian populations, and a person homozygous for the codon 972 mutation responded diabetically to a dexamethasone challenge. [56] Pima Indians do not carry the polymorphism G972R. A number of in vitro tests have been conducted to investigate the molecular effects of the G972R polymorphism because it is linked to type 2 diabetes, sits between two putative tyrosine phosphorylation sites that are involved in the binding of the p85 subunit of PI3-kinase, and may disrupt PI3-kinase’s binding to IRS-1. In fact, the expression of the G972R variation of IRS-1 in 32D(IR) cells results in a 36 percent reduction in IRS-1–associated PI3-kinase activity as well as a specific deficiency in the binding of the p85 subunit of PI3-kinase to IRS-1. The notion that G972R disrupts the connection between IRS-1 and the SH2 domains of PI3-kinase is supported by the fact that insulin-stimulated IRS-1 tyrosine phosphorylation is normal. As a result, the ultimate biological action of insulin in these cells is reduced by 35–40%: Mitogenesis stimulation [57]. The p85alpha subunit of PI3-kinase frequently undergoes a polymorphism that converts methionine at position 326 to isoleucine. The mutation was found to be heterozygous in 31% of Caucasians and homozygous in 2% of them in one investigation. The functional effects of this polymorphism, which lies between the SH3 and first SH2 domains, have not been investigated in vitro. In an intravenous glucose tolerance test, homozygous people show a 32% decrease in insulin sensitivity when compared to wild-type and heterozygous carriers, even though the frequency is not elevated in diabetes [58]. Peroxisome proliferator–activated receptor gamma (PPAR-γ) One nuclear receptor that seems to be a key modulator of adipogenesis is PPAR-γ. There are two isoforms of PPAR-γ: γ1 and γ2. While PPAR-γ2 is primarily expressed in adipose tissue, PPAR-γ1 is present everywhere. Insulin sensitizers like troglitazone and pioglitazone bind to PPAR-γ with high affinity, triggering the regulation of gene transcription even though PPAR-γ is not directly a component of the insulin signaling pathway. Constitutively active PPAR-γ is caused by a rare, naturally occurring mutation of PPAR-γ (P115Q) in the close vicinity of a significant regulatory phosphorylation site. PPAR-γ 2 (P12A) was linked to a lower body mass index and better insulin sensitivity [59]. Extreme obesity without noticeable insulin resistance is a symptom of this mutation. P115Q overexpression in 3T3-L1 fibroblasts causes them to quickly differentiate into adipocytes in vitro. Thus, a constitutively active form of PPAR-γ causes this uncommon type of obesity by increasing adipogenesis while also making the entire body more sensitive to the effects of insulin. Remarkably, it was recently discovered that a different mutation in a distinct area. Losing weight enhances insulin sensitivity via restoring tyrosine kinase activity and insulin receptor expression. However, the partial recovery of insulin sensitivity in type 2 diabetes raises the possibility of a further post-receptor malfunction of insulin signaling. The liver’s insulin receptor count is reduced in animal models of hereditary and acquired obesity, and this can be fixed by reducing hyperinsulinemia.[60] These findings imply that obesity, or more likely, the accompanying hyperinsulinemia, is the primary cause of the downregulation of insulin receptor expression and tyrosine kinase activity. However, several other processes, such as modifications in membrane lipids brought on by hyperlipidemia, may also play a role in the insulin resistance linked to obesity, in addition to decreased insulin receptor expression and tyrosine kinase activity.[61] Tumor necrosis factor-alpha TNF–α TNF-alpha was initially shown to be an endogenous cytokine generated by lymphocytes and macrophages during inflammatory activation. Chromosome 6 contains the pro-inflammatory cytokine TNF-α (6p21). [62] Numerous cells, including macrophages, immune T cells, immunological β-cells, as well as osteoblasts, epithelial, smooth muscle, and tumor cells, can produce TNF-α [63]. Like the majority of peptide effectors, TNF-alpha is thought to work via transmembrane receptors. TNF-R1 (p55 in rodents and p60 in humans) is the name of the two TNF receptors that have been discovered.[64] and TNF-R2 (p80 in humans, p75 in rodents) are co-expressed in almost all cells, though in varying proportions. [65] Both of these receptors can bind to TNF-α as well as LT-α, a similar protein that is also known as TNF-fJ. There is a significant overlap in the functions of TNF-α and β, as would be predicted when ligands engage overlapping receptors. However, outside of the ligand binding region, the two TNF receptors seldom resemble one another, indicating that they signal for distinct biological processes. [66] It was shown that TNF-α suppressed insulin receptor (IR) signaling by phosphorylating serine residues present in the insulin receptor IRS substrate. [67] In one of these models, TNF-α neutralization increases insulin receptor tyrosine kinase activity, particularly in muscle and fat tissues, improving insulin sensitivity. TNF-α is a strong cellular inhibitor of insulin-stimulated tyrosine phosphorylation on the 3-chain of the insulin receptor and insulin receptor substrate-1, indicating a malfunction at or close to the insulin receptor’s tyrosine kinase activity. These findings strongly imply that TNF-α may be a key factor in the systemic insulin resistance of NIDDM, given the obvious connection between obesity, insulin resistance, and diabetes.[68] TNF-α is now known to define a family of cytokine effectors that modulate a wide range of immune functions.[69] Similar to other cytokines, TNF-α serves a wide range of purposes in the immune and extra-immune systems. Apoptotic cell death induction, tumor cell lysis, thymocyte proliferation, stimulation of other cytokine production, including granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-1 (IL-1), and suppression of lipoprotein lipase activity are a few examples.[70] The transcription process of the TNF-α gene can be accelerated by substituting the nucleotide G for A at position -308 in the promoter region. Strong genome transcription raises TNF-α production, which raises the incidence of type 2 diabetes.[71] When applied at relatively high doses, TNF-α suppresses the majority of lipogenic enzymes, including lipoprotein lipase, and causes adipocytes to ”de-differentiate.”[72] Since the absolute levels of insulin-stimulated glucose disposal in the obese animals do not equal those in their lean littermates, the increase in insulin sensitivity following TNF-α neutralization represents a significant improvement but not a total reversal. To fully understand the role of these cytokines in obesity-related insulin resistance at pertinent sites of insulin action, more experimental systems will be required.[73] Two potentially relevant systems—GLUT-4 and the insulin receptor itself—have demonstrated direct effects of TNF-α on insulin-sensitive cells. GLUT4 mRNA levels in adipocytes and myocyte cultures have been demonstrated to be downregulated by TNF-α. [74] In these investigations, the actual TNF-α-induced defect is probably at or close to the insulin receptor. Reduced autophosphorylation is observed in partially purified receptors isolated from TNF-α-treated cells, and in these investigations, the phosphorylation of exogenously added recombinant induced by TNF-α is probably at or close to the insulin receptor itself. TNF-α-treated cells’ partially purified receptors exhibit decreased autophosphorylation and phosphorylation of exogenously added recombinant.[75] GLUT4 mRNA levels were not significantly impacted by TNF-α neutralization. However, in animals pretreated with the TNFR-IgG fusion as opposed to vehicle alone, the tyrosine phosphorylation of the insulin receptor and IRS-1 was significantly elevated in response to acute insulin injection. Fat and muscle showed this effect, but the liver showed no signs of it. Following TNFR-IgG injection, there were no changes in the absolute levels of the insulin receptor or IRS-1 protein, suggesting that the agent’s action was limited to particular amounts of tyrosine phosphorylation per protein molecule. Tyrosine phosphorylation was elevated in every animal that received the neutralizing agent, and in certain instances, the levels were comparable to the insulin-stimulated phosphorylation of the insulin receptor or IRS-1 seen in lean control animals.[76] One important question is whether TNF-α suppresses insulin function in muscle and fat directly or indirectly. The mRNA for Type I and Type II TNF receptors, which have been identified, is expressed in both tissues. Since adipose tissue produces TNF-α mRNA and protein, and because exogenous TNF-α has been demonstrated to inhibit insulin action in adipocytes, it is reasonable to assume that this cytokine acts directly in fat cells through an autocrine loop.[77] Delineating the precise molecular mechanisms by which TNF-α inhibits insulin signaling will be crucial. Given that the partially purified insulin receptor from TNF-α-treated cells shows reduced kinase activity, it is plausible that the insulin receptor is covalently modified in these cells.[78] For instance, it has been demonstrated that decreased tyrosine kinase activity correlates with covalent modification of insulin receptors through serine and threonine phosphorylation, and that TNF-α can induce multiple protein kinases in specific cellular systems. It has also recently been shown that serine/threonine phosphorylation of IRS-1 can disrupt the action of insulin.[79] Investigations into these possibilities are ongoing. Another possibility is that a tyrosine phosphatase or a co-purifying inhibitory molecule is the cause of the decreased kinase activity. [80] Purification and characterization of these molecules will be crucial if such a protein or activity is seen after TNF-α treatment. Whether or not TNF-alpha is the physiological effector in humans, knowledge of the signal transduction pathways that obstruct insulin action may be broadly relevant in NIDDM. This implies that either TNF-alpha stimulates the synthesis of a receptor inhibitor linked to these preparations or that the insulin receptor itself is altered.[81] Last but not least, TNF-alpha’s impact on insulin receptor signaling might not be totally unique to TNF; when applied to intact cells, IL-1 and IL-6 also lessen insulin receptor auto-phosphorylation and IRS-1 tyrosine phosphorylation.[82] This effect is not totally specific because it happens in fat cells when the expression of several genes specific to fat, like adipsin or aP2, is downregulated. However, GLUT4 is not the only gene that exhibits aberrant expression in obesity; adipsin is also down-regulated in many animal models of obesity, though this has not yet been noted in human research. Furthermore, at doses that do not affect the cellular content of GLUT4 protein, TNF-alpha can significantly inhibit insulin-stimulated glucose transport. Therefore, it is unlikely that the glucose transporter level alone is the mechanism underlying TNF-α-induced insulin resistance in cultured cells.[83] Adipose tissue is one of the many cell types that express TNF-α. Weight loss reduces TNF-alpha expression, while obese animals’ and humans’ adipocytes overexpress TNF-α in positive correlation with body mass index and hyperinsulinemia.[84] Obesity is likely to increase local concentrations of both free and membrane-bound TNF-α, even though this local release of TNF-α has little effect on systemic TNF-α concentrations. By raising the serine phosphorylation of IRS-1 (and potentially other IRS proteins), TNF-α seems to disrupt insulin signaling. Insulin receptor tyrosine kinase activity is inhibited by serine-phosphorylated IRS-1, impairing downstream signaling. It has been proposed that an additional insulin receptor inhibitory factor (perhaps a tyrosine phosphatase or an inhibitor of serine phosphatases) binds to IRS-1 as a result of TNF-α-induced serine phosphorylation of IRS-1, mediating the inhibition of the insulin receptor kinase. Mice with a targeted mutation of both TNF-α receptor isoforms, p55 and p75, exhibit improved insulin sensitivity due to a complete lack of TNF-α signaling. It seems that the p55 receptor isoform has a greater effect. Targeted disruption of multiple insulins signaling proteins, including the insulin receptor, IRS-1, and IRS-2, has been one strategy. In homozygous animals, targeted disruption of the insulin receptor results in death within a few days of birth, while heterozygous animals exhibit virtually no phenotype. IGF-1 resistance causes growth retardation when IRS-1 is disrupted, but mild insulin resistance and impaired glucose tolerance without diabetes are the only side effects. Insulin resistance is also caused by IRS-2 disruption, but in this instance, there is also a decrease in β-cell mass, which results in diabetes. More severe insulin resistance and a phenotype that resembles human type 2 diabetes, including a delayed onset of diabetes, result from the combination of heterozygous insulin receptor knockout and heterozygous IRS-1 knockout. This type of insulin resistance is characterized by high insulin levels. It’s interesting to note that only 40% of these mice get diabetes, indicating the significance of other background genes. Discussion Following insulin’s binding to its receptor, certain and tightly controlled processes take place. While identifying the critical processes that contribute to insulin signaling specificity is a significant challenge for biochemical research, the results should provide novel therapeutic strategies for the management of patients with insulin-resistant conditions, such as type 2 diabetes.[85] Both humans and rodents with insulin resistance have shown increased IR serine phosphorylation, which is linked to decreased tyrosine kinase activity.[86] While there are several sites where inhibitory IRS-1 serine phosphorylation takes place [30], the most well-studied of these modifications is at Ser-307. It is generally accepted that this contributes to insulin resistance by blocking insulin receptor kinase activity, but recent research has cast doubt on this association. Insulin itself can trigger the phosphorylation of IRS-1 on Ser-307 in both humans and mice; a knockout of the IRS-1 Ser307Ala mutant developed more severe insulin resistance than control mice when given a high-fat diet, suggesting that Ser-307 is necessary to maintain normal insulin signaling. [88] Therefore, increased IRS-1 Ser-307 phosphorylation observed in insulin-resistance states may be linked to, but not the cause of, insulin resistance. While IRS2 gene knockout animals have insulin deficiency primarily in the liver, no effect on cell differentiation, and produce growth abnormalities in a few tissues, including neurons and pancreatic cells, IRS1 gene knockout mice cause abnormal cell differentiation in preadipocytes and show insulin resistance in muscle tissue.[89] The IRS protein’s PTB-domain has an NPXpY-recognition motif created by phosphotyrosine at site 960 of the beta-subunit, which is located just inside the membranes. When this tyrosine is altered, the majority of insulin-dependent biological activities are lost, and subsequent phosphorylation of IRS-1 and other insulin receptor substrates is totally inhibited.[16] More severe insulin resistance and a phenotype that resembles human type 2 diabetes, including a delayed onset of diabetes, result from the combination of heterozygous insulin receptor knockout and heterozygous IRS-1 knockout. This type of insulin resistance is characterized by high insulin levels. It’s interesting to note that only 40% of these mice get diabetes, indicating the significance of other background genes. Elevated levels of serine/threonine phosphorylation in IRS1 decrease IRS1 affinity with the p85 regulatory subunit of PI3K, weakening insulin signal transduction and resulting in the symptom of insulin resistance. Additionally, IRS1 phosphorylation at Ser636/639 and Ser307 prevents IRS1 from binding to IR. [90] Insulin receptor kinase activity is negatively regulated by PTPs and LAR. The elevated expression and activity of various protein tyrosine phosphatases (PTPs), which dephosphorylate and thereby stop signaling propagated through tyrosine phosphorylation events, may be one mechanism for the signaling defects in obesity.[91] According to certain data, the muscle and adipose tissue of obese humans and rodents exhibit elevated expression and/or activity of at least three PTPs, including PTP1B, leukocyte antigen–related phosphatase (LAR), and Src-homology-phosphatase 2. In vitro, it has been demonstrated that PTP-1B and LAR dephosphorylate IRS-1 and the insulin receptor. Due in part to increased energy expenditure, mice with PTP-1B knockout exhibit increased insulin sensitivity and resistance to diet-induced obesity. This implies that PTP-1B regulates both insulin action and energy homeostasis.[92] Other proteins, including growth-factor-receptor-bound protein 10 (Grb10), plasma-cell-membrane glycoprotein-1 PC-1, and suppressor of cytokines signaling-1 (SOCS1 and SOCS3), inhibit IR function by either altering its kinase activity or sterically blocking its interaction with the IRS proteins. By encouraging insulin receptor catalytic activity and preventing tyrosine dephosphorylation of IRS proteins, Sh2-B1 directly binds to insulin receptors and IRS proteins, improving insulin sensitivity.[93] The two subunits of PI3-K, the catalytic and regulatory subunits, are in charge of phosphorylating PIP2 to PIP3 in the cellular membrane. Unexpectedly, increased insulin sensitivity is seen in all PI3K regulatory subunit knockouts, including p85a heterozygous deletion, p85b KO, and p50a/p55a double KO.[94] There are several known ways to enhance insulin action by decreasing the concentration of regulatory subunits. The enzymatically competent p85/p110 heterodimer competes with regulatory subunits for binding to IRS proteins because they are usually present in higher concentrations than catalytic subunits. The regulation of the phosphatase and tensin homolog (PTEN) has also been connected to the p85a monomer.[95] More recently, it was shown that p85a changes the unfolded protein response and interacts with the transcription factor XBP-1, both of which are implicated in insulin resistance.[96] It is still not known that if IRS-3 and IRS-4 are in any way involved in insulin and IGF signaling, because KO of these two molecules does not produce any observable phenotype.[97] The different phenotypes that result from the KO of the two receptors or their substrates show that each receptor requires many substrates to mediate its functions.[98] Although serine phosphorylation of IRS1 is strongly associated with insulin resistance, its exact role in the pathogenesis of insulin resistance is yet unknown. Moreover, it is currently unknown if serine phosphorylation of additional IRS proteins might have a substantial regulatory role.[99] Transcription factors from the Forkhead box O (Foxo) family control the expression of gluconeogenic and lipogenic genes. Multiple Akt phosphorylation sites on Foxos provide docking sites for binding proteins of the 14-3-3 family. This interaction results in Foxo’s exclusion from the nucleus by blocking its transcriptional activity.[100] Interestingly, animals with Akt1 and Akt2 impairment show significant hepatic insulin resistance and high levels of hepatic glucose production. These abnormalities are eliminated when Foxo1 is ablated in the liver at the same time. This implies that insulin-mediated control of hepatic glucose production may also be involved in the regulation of hepatic glucose metabolism in addition to the Akt/Foxo1 axis is not the sole mechanism that controls the metabolism of glucose in the liver; insulin can also control the synthesis of glucose in the liver. Because Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS), which catalyzes the manufacture of the vasodilator and anti-inflammatory chemical nitric oxide (NO), there may be a link between insulin resistance and cardiovascular disease.[101] Protein phosphatase 2A (PP2A) is responsible for 80% of serine/threonine phosphatase activity in cells. It also regulates the activities of many protein kinases implicated in insulin action, including Akt, PKC, S6K, ERK, cyclin-dependent kinases, and IKK.[102] Numerous investigations have shown that PP2-A is hyperactivated in diabetes situations.[103] PHLPP-1 and -2 are leucine-rich repeat protein phosphatases with a PH domain. which dephosphorylate both Akt and PKCs, are two new members of the PP2-C family that regulate the action of insulin.[104] Reduced glycogen synthesis and glucose transport are the results of PHLPP-1 overexpression in cells, which also affects Akt and glycogen synthase kinase 3 activity.[105] Patients with diabetes and/or obesity have higher levels of PHLPP-1 in their skeletal muscle and adipose tissue, which is correlated with lower Akt2 phosphorylation.[106] Mice with whole-body PTEN haploinsufficiency exhibit enhanced insulin sensitivity and improved glucose tolerance, while mice with muscle, adipose tissue, or liver-specific PTEN deletion have higher insulin sensitivity. It has recently been shown that the regulatory subunit of PI3-K, which is p85a, binds directly to PTEN and enhances its activity, establishing a special link between PIP3 synthesis and degradation.[107] Through their metabolic product diacylglycerols, lipids can activate both classical (α,β,γ) and new PKC members (δ,ε,θ) and disrupt insulin signaling by causing multiple serine phosphorylation of IRS proteins and IR, particularly at Thr-1336, Thr-1348, and Ser-1305/1306.[108] Therefore, deletion of any member of the new PKC family prevents the development of insulin resistance in the skeletal muscle and liver via decreasing IRS-1 Ser-307 phosphorylation.[109] PPAR-γ is one nuclear receptor that appears to be an important adipogenesis regulator. PPAR-gamma comes in two isoforms: γ1 and γ2. PPAR-γ1 is found everywhere, but PPAR-γ2 is mostly expressed in adipose tissue. Insulin sensitizers like troglitazone and pioglitazone bind to PPAR-γ with high affinity, modifying gene transcription even though they are not a direct part of the insulin signaling system. Constitutively active PPAR-γ is caused by a rare, naturally occurring mutation of PPAR-γ (P115Q) close to a crucial regulatory phosphorylation site. Extreme obesity without noticeable insulin resistance is the result of this mutation.[110] Based on a study conducted on 3T3-L1 mice fibroblasts shows that they instantly differentiate into adipocytes when the P115Q mutation is overexpressed. Therefore, through PPAR-γ, stimulating adipogenesis and increasing the body’s sensitivity to the effects of insulin, a constitutively active form of PPAR-γ causes this rare form of obesity.[111] Alternative splicing of the PI3-kinase regulatory subunits in the liver is one of the other changes in the insulin signaling cascade observed in hyperinsulinemic models of obesity.[112] Both Zucker fatty rats and leptin-deficient ob./ob. mice exhibit downregulation of p85a and a twofold increase in p50a expression. While AS53 is downregulated by about 50 in Zucker fatty rats, it is dramatically upregulated in the liver of ob./ob. mice. This could have an impact on how the insulin signal is routed or compartmentalized in these models. It is unknown if alterations in PI3-kinase regulatory subunit alternative splicing also take place in human obesity.[113] As a result, while Akt1 and Akt3 KO mice do not develop diabetes, Akt2 KO mice do. A constitutively active PPAR-γ is produced by a rare, naturally occurring mutation of PPAR-γ(P115Q) close to a crucial regulatory phosphorylation site. Extreme obesity without noticeable insulin resistance is a symptom of this mutation.[53] In one of these models, TNF-α neutralization increases insulin receptor tyrosine kinase activity, particularly in muscle and fat tissues, improving insulin sensitivity. Mice with a targeted mutation of both TNF-α receptor isoforms, p55 and p75, exhibit improved insulin sensitivity due to a complete lack of TNF-α signaling. It seems that the p55 receptor isoform has a greater effect. Conclusion We recently showed that mice, known as L-DKO mice (liver double IRS1 and IRS2 gene knockout mice), developed hyperglycemia, hyperinsulinemia, insulin resistance, and hypolipidemia as a result of having both IRS1 and IRS2 genes deleted in their livers. This prevented the activation of hepatic Akt/Foxo1 phosphorylation. When both IRS 1 and IRS 2 were deleted from the heart muscle, the phosphorylation of Foxo1 (S253) and Akt (T308 and S473) was reduced, and male animals that were 6–8 weeks old died suddenly. These findings suggest that insulin resistance and heart failure may be significantly influenced by the loss of IRS 1 and IRS 2. PTP-1B and LAR are negative regulators of insulin receptor kinase activity, as was discussed, but obesity-related overexpression results in insulin resistance. Due in part to increased energy expenditure, the gene that produces these proteins in KO mice can increase insulin sensitivity and resistance to diet-induced obesity. It’s interesting to note that adipocytes lack insulin sensitivity, but muscle and liver do. One important question that needs to be addressed is whether insulin sensitivity and leanness/energy expenditure are causally related or if they are controlled by different signaling pathways. TNF-α Neutralization Increased TNF-alpha was linked to obesity and insulin resistance. The overexpression of Ser/Thr phosphorylation activation by these cytokines inhibits further signaling and IR-IRS phosphorylation. One important topic is whether TNF-alpha directly or indirectly inhibits insulin action in muscle and fat. Both organs exhibit expression of the recognized Type I and Type II TNF receptor mRNA. It makes sense to believe that TNF-alpha operates directly in fat cells via an autocrine loop since adipose tissue produces both the mRNA and the protein, and exogenous TNF-alpha has been shown to suppress insulin action in adipocytes. The increase in insulin sensitivity after TNF-alpha neutralization is a considerable improvement, but not a complete regression, because the obese animals’ absolute levels of enhanced stimulated glucose disposal do not match those of their lean littermates. Because TNF-alpha signaling is completely absent in mice with a specific mutation of both TNF-alpha receptor isoforms, P55 and p75, the mice have increased insulin sensitivity. The p55 receptor isoform appears to have a stronger impact. Additional experimental systems will be needed to completely comprehend the involvement of this cytokine in obesity-associated insulin resistance at relevant locations of insulin action. What is the extent to which TNF-α contributes to insulin resistance, and why does its suppression only partially restore insulin sensitivity in some models? What specific serine kinases are activated in insulin-resistant situations, and how do they connect to IRS-1’s activity through serine phosphorylation? What link is there between mitochondrial dysfunction and the activation of these serine kinases? PI3-K catalytic activity. PI3K regulatory subunit knockouts, including p85a heterozygous deletion, p85b KO, and p50a/p55a double KO, are characterized by increased insulin sensitivity. By lowering the concentration of regulatory subunits, insulin action can be improved in a number of ways. The enzymatically competent p85/p110 heterodimer competes with regulatory subunits for binding to IRS proteins because they are usually present in higher concentrations than catalytic subunits. The control of the phosphatase and tensin homolog (PTEN) has also been connected to the p85a monomer. What role does elevated PI3K p85α regulatory subunit expression play in impaired insulin signaling in insulin resistance? Does clinically significant insulin resistance require both serine-phosphorylated IRS-1 and elevated p85α? Systemic Insulin Sensitivity and Adipocyte GLUT4: The expression of GLUT4 in adipocytes is essential for preserving insulin responsiveness throughout the body. It is still unknown whether impaired glucose transport in fat causes systemic insulin resistance directly or indirectly through changed adipocyte secretion of signaling molecules. Selective GLUT4 modulation models should be used in future studies to isolate these effects. PPAR-γ(P115Q) mutation A constitutively active PPAR-γ is produced by a rare, naturally occurring mutation of PPAR-γ(P115Q) close to a crucial regulatory phosphorylation site. Extreme obesity without noticeable insulin resistance is a symptom of this mutation. More research is required to determine whether obesity in this instance affects insulin sensitivity for therapeutic reasons. With obesity, PPAR-γ can raise insulin sensitivity. P115Q overexpression in 3T3-L1 fibroblasts causes them to quickly differentiate into adipocytes in vitro. Thus, a constitutively active form of PPAR-γ causes this uncommon type of obesity by increasing adipogenesis while also making the entire body more sensitive to the effects of insulin. It’s interesting to note that a different mutation in the distinct PPAR-γ2 region (P12A) was recently linked to better insulin sensitivity and a lower body mass index. References [1] Cullen M Taniguchi, Brice Emanuelli, and C Ronald Kahn. Critical nodes in signalling pathways: insights into insulin action. Nature Reviews Molecular Cell biology, 7(2):85–96, 2006. [2] Antti VirkamÅNaki, Kohjiro Ueki, C Ronald Kahn, et al. Protein–protein Interaction in insulin signaling and the molecular mechanisms of insulin resistance. The Journal of clinical investigation, 103(7):931–943, 1999. [3] Lei Sun, Huangming Xie, Marcelo A Mori, Ryan Alexander, Bingbing Yuan, Shilpa M Hattangadi, Qingqing Liu, C Ronald Kahn, and Harvey F Lodish. Mir193b–365 is essential for brown fat differentiation. 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Authors Affiliations Syed Sohail Ahmad 0009-0005-5129-3864 [email protected] University of Malakand View all articles by this author Metrics & Citations Metrics Article Usage 1861 views 213 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Syed Sohail Ahmad. Impairment of the IR-IRS-PI3K-AKT signaling pathway in Insulin Resistance: New Insights into Insulin Resistance Mechanisms. Authorea . 12 September 2025. DOI: https://doi.org/10.22541/au.175767205.55981786/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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