Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2.

“In contrast, ‘priming’ phosphorylation of AKT at S473—by mammalian target of rapamycin (mTOR) complex 2 (mTORC2) [21] (Fig.”

“phosphorylation is frequently used as evidence of insulin resistance (or its resolution) in mice, despite the influence of insulin on S307 phosphorylation and the probable contribution of other phospho-S/Ts [44].”

“Increased plasma BCAA levels in obese, insulin-resistant persons have long been regarded as part of an insulin-resistant metabolic ‘signature’ [110, 111], but supplementation of rodent diets with mixed BCAA or leucine has not clarified that this is diabetogenic [111, 112].”

“Prospective studies first identify insulin resistance in human skeletal muscle, which is thought to contribute, via a dys-metabolic process, to the emergence of phenotypic disease [3, 74].”

“but not Akt1, gene deletion in mice causes a diabetes-like phenotype [22].”

“1b); S6K2 can also phosphorylate IRS1 in vitro, but does not compensate for loss of S6K1 in knockout mice [51, 106].”

“Recruitment of dimeric PI3K (p85•p110) to IRS produces membrane phosphatidylinositol 3,4,5-trisphosphate (PIP3), which recruits the S/T kinase AKT [19].”

“IKKβ and JNK—Impaired glucose uptake and/or insulin resistance within adipose tissue can contribute to the development of type 2 diabetes [164], likely by altering its secretion of adipokines, such as adiponectin or retinol-binding protein 4 (reviewed in [165]).”

“NEFA levels causes insulin resistance in rats, accompanied by elevated muscle IRS1 S307 (hS312) phosphorylation [146].”

“Treatment of CHO cells with the low-specificity JNK agonist anisomycin was used to identify S307 (hS312) near the IRS1 PTB domain as a major site of phosphorylation [38].”

“Lipid infusion in humans can also activate muscle PKCδ and (canonical) PKCβ2 [154].”

“The activation/levels of two other IRS kinases, ROCK1 and G protein-coupled receptor kinase 2 (GRK2), is mechanistically independent of IRS [20] but is clearly influenced by insulin and altered in type 2 diabetes [88–90].”

“Intramyocellular diacylglycerol can be generated in humans and rodents by normal phospholipid metabolism or, experimentally, by the infusion of TAG emulsions plus heparin to activate tissue lipase [153].”

“Additionally, the expanded adipose compartment in diabetes/obesity produces proinflammatory cytokines such as TNFα and IL1β that can contribute to insulin resistance systemically (reviewed in [79]).”

“intramyocellular TAG , hepatosteatosis) (reviewed in [74]).”

“In contrast with AKT, basal or insulin-stimulated ERK1/2 activation in diabetic muscle is unchanged or increased in several studies, compatible with its partial independence from IR/IRS [82, 86].”

“It can now be appreciated that feedback S/T phosphorylation of IRS by mTORC1 and/or S6K1 underlies their disappearance during persistent insulin stimulation [49, 50].”

“Related pathways that contribute to the regulation of AKT include: (1) mTORC1 phosphor-ylation/ stabilisation of GRB10 [174], which inhibits productive interaction of IRS with a key phosphotyrosine in IR [175]; (2) inhibition of mTORC2 by S6K1-mediated phosphorylation of rictor [176]; and (3) transcriptional upregulat…”

“In cultured cells that produce very little IRS2, insulin stimulates S307 phosphorylation via a PI3K pathway requiring the IRS1 YXXM motifs [39, 40].”

“Given their co-incidence and propensity to aggravate one another, it cannot be stated whether hyperinsulinaemia or dyslipidaemia (or still other factors) underlie increased IRS S/T phosphorylation in the pre-diabetic period [85].”

“in all tissues, exhibit abnormal or severely compromised adipocyte differentiation [16, 17].”

“It can now be appreciated that feedback S/T phosphorylation of IRS by mTORC1 and/or S6K1 underlies their disappearance during persistent insulin stimulation [49, 50].”

“In contrast, in mammalian cells, IKKβ physically associates with IRS1, and its TNFα-stimulated activity correlates with phosphorylation of mS307/hS312—consistent with IKKβ phosphorylation of this residue in vitro [167].”

“IKKβ and JNK—Impaired glucose uptake and/or insulin resistance within adipose tissue can contribute to the development of type 2 diabetes [164], likely by altering its secretion of adipokines, such as adiponectin or retinol-binding protein 4 (reviewed in [165]).”

“Thus, though AKT signalling to mTORC1 is diminished in diabetes, the over-supply of nutrients in obesity is hypothesised to promote mTORC1/S6K phosphorylation of IRS and other targets, aggravating insulin resistance (reviewed in [109, 110]).”

“IRS2 (but not IRS1) protein concentration in mouse liver is decreased during refeeding via a PI3K-dependent mechanism [68].”

“It also underlies risk factors—including hyperglycaemia, dyslipidaemia and hypertension—for the clustering of type 2 diabetes with cardiovascular disease, non-alcoholic fatty liver disease and related maladies (metabolic syndrome) [2].”

“Increased plasma BCAA levels in obese, insulin-resistant persons have long been regarded as part of an insulin-resistant metabolic ‘signature’ [110, 111], but supplementation of rodent diets with mixed BCAA or leucine has not clarified that this is diabetogenic [111, 112].”

“TNFα activates multiple kinases in muscle, including S6K, ERK1/2, IKKβ and the IRS1 mS307/hS312 kinase JNK1; however, muscle hS312 phosphorylation is not significantly elevated by TNFα infusion prior to the period in which TNFα causes reduced IRS1 tyrosine phosphorylation and insulin resistance [34].”

“Other agents—including insulin in rodent and human tissues—can stimulate phosphorylation of S307 via JNK-independent pathways [39].”

“Thus, S/T phosphorylation at many sites together converts IRS proteins to an inactive state, separable from their inactivation by degradation; it was further suggested that this ‘array phosphorylation’ of IRS (particularly within the PTB-proximal regions) is necessary for the desensitisation of insulin signalling by d…”

“Comparison of knock-in mice with constitutively active GSK3 isoforms (α, β, or α and β) demonstrates the predominance of GSK3β in muscle, yet none of these mice show impaired AKT activation or become diabetic [140].”

“Rodents fed a high-fat diet to cause obesity and insulin resistance show increased basal mTORC1→S6K activation in metabolic tissues, with pronounced phosphorylation of IRS1 at S632 and/or S302 (hS636, hS307) [106, 115].”

“The correlation between excessive β-adrenergic activity and insulin resistance has long been noted [131].”

“Insulin resistance, defined as reduced responsiveness of tissues to normal insulin concentrations, is a principal feature of type 2 diabetes, which leads to compensatory hyperinsulinaemia [1].”

“5 mmol/l NEFA) impairs muscle IRS1 tyrosine phosphorylation, PI3K activity and glucose disposal, and these effects are lost in Prkcq (PKCθ) knockout mice [147].”

“Rodent and human studies validate the contribution of dyslipidaemia to skeletal muscle and liver insulin resistance via diacylglycerol-mediated activation of nPKCs, including PKCθ [65, 146, 147], PKCδ [148] and PKCε [149, 150] (reviewed in [74, 94]).”

“Nonetheless, mTORC1/S6K can be stimulated by nutrients independently of IRS [107]—most clearly by branched-chain amino acids (BCAA) such as leucine [108].”

“Increased plasma concentration of the vasoconstrictive ET-1 polypeptide is associated with insulin resistance and/or hypertension [128, 129], which is, in turn, promoted by direct and indirect (sympathoadrenal and angiotensin II-dependent) effects of compensatory hyperinsulinaemia to cause sodium retention (reviewed i…”

“Mice lacking mTOR or raptor in muscle, or S6K1 in all tissues, highlight the role of mTORC1→S6K signalling in the growth and maintenance of skeletal muscle (reviewed in [107]).”

“Insulin stimulation of the RAS→mitogen-activated protein kinase (MAPK) cascade depends partly on other substrates of the IR, such as SHC, yet activation of the MAPKs extracellular signal-regulated kinase-1 and -2 (ERK1/2) is impaired in IRSdeficient mouse liver [11].”

“The binding of insulin to the insulin receptor (IR), and in muscle tissues to hybrid insulin/ IGF1 receptors (IR/IGF1R) [7], promotes the autophosphorylation of a trio of regulatory loop tyrosine residues, disinhibiting IR tyrosine kinase activity toward IRS [8].”

“mTOR complex 1 (mTORC1) can mediate insulin- or anisomycin-stimulated S307 phosphor-ylation [41], casting some doubt on the significance of insulin-stimulated JNK phosphorylation of S307 observed in cultured cells [42].”

“However, as reviewed herein and elsewhere [5, 6], unique phospho-S/T on IRS1 can have negative (or positive) effects on insulin signalling that are currently unexplainable in either structural terms or by a requirement for multiple phosphorylation.”

“Related pathways that contribute to the regulation of AKT include: (1) mTORC1 phosphorylation/ stabilisation of GRB10 [174], which inhibits productive interaction of IRS with a key phosphotyrosine in IR [175]; (2) inhibition of mTORC2 by S6K1-mediated phosphorylation of rictor [176]; and (3) transcriptional upregulati…”

“However, SIK2 kinase activity is positively regulated by insulin via phosphorylation by AKT2 [144], suggesting an autologous feedback role in the regulation of IRS.”

“Surprisingly—given the apparent involvement of phospho-S632 (hS636) in desensitisation of insulin signalling [53, 91]—double mutation of S632/ 635 to alanine in CHO cells reduces IRS1 tyrosine phosphorylation and PI3K binding [71].”

“In contrast, infusion of complex amino acid mixtures (AA) into humans with clamped hyperinsulinaemia (to repress gluconeogenesis from amino acid precursors) decreases the rate of glucose disposal by about a third, with post-clamp muscle biopsies showing decreased IRS1 tyrosine phosphorylation and increased phosphoryla…”

“The binding of insulin to the insulin receptor (IR), and in muscle tissues to hybrid insulin/ IGF1 receptors (IR/IGF1R) [7], promotes the autophosphorylation of a trio of regulatory loop tyrosine residues, disinhibiting IR tyrosine kinase activity toward IRS [8].”

“Potentially contributing to the latter finding, ERK1/2 can promote lipolysis and NEFA-mediated insulin resistance, both by downregulating adipocyte perilipin [120] and activating hormone-sensitive lipase [121].”

“Of these, phospho-S312, -S616, -S636 and -S1101 are associated with desensitisation of insulin signalling (above and [56, 65]), whereas phospho-S1223 has a sensitising effect [31].”

“Solinas G, Karin M. JNK1 and IKKβ: molecular links between obesity and metabolic dysfunction.”

“Nonetheless, mTORC1/S6K can be stimulated by nutrients independently of IRS [107]—most clearly by branched-chain amino acids (BCAA) such as leucine [108].”

“Related studies show that decreased muscle glucose transport via the GLUT4 path underlies this alteration [74, 77].”

“The PH domain is the principal module that links IRS1 with the activated IR [26], whereas the PTB domain enhances this interaction via binding to the phospho-NPEY motif within the IR juxtamembrane region [27].”

“Notably, in mouse embryonic fibroblasts, IKKβ is required for TNFα-stimulated phosphorylation of IRS1 S265; mutation of this residue in HEK293 cells (hS270) reduces TNFα stimulation of IRS1–S6K interaction and other IRS1 phospho-S/T elevated in adipose tissue from ob/ob mice [55].”

“Increased plasma concentration of the vasoconstrictive ET-1 polypeptide is associated with insulin resistance and/or hypertension [128, 129], which is, in turn, promoted by direct and indirect (sympathoadrenal and angiotensin II-dependent) effects of compensatory hyperinsulinaemia to cause sodium retention (reviewed i…”

“upstream insulin resistance), the implication is that mTORC1 activity is reduced in insulinresistant human adipocytes in vivo [116].”

“Increased plasma BCAA levels in obese, insulin-resistant persons have long been regarded as part of an insulin-resistant metabolic ‘signature’ [110, 111], but supplementation of rodent diets with mixed BCAA or leucine has not clarified that this is diabetogenic [111, 112].”

“AKT may also phosphorylate S267 (hS272) in TNFα-treated cells [105].”

“For example, the simultaneous mutation to alanine of four S/Ts near IRS1 YXXMs (mS612/632/662/731A; hS616/636/ 666/736A) was shown to increase basal and insulinstimulated IRS1 tyrosine phosphorylation and associated PI3K activity [33].”

“Prior treatment with proteasome inhibitors can block the insulin-induced degradation of IRS1, but not its intracellular transit or the desensitisation of downstream signalling and glucose uptake [48].”

“Mutation of S612 is sufficient to protect HEK293 cells against the desensitisation of insulin signalling by phorbol 12-myristate 13-acetate (PMA)-activated novel and conventional PKC (cPKC) isoforms [155].”

“Any regulatory effects of AKT-mediated IRS phosphorylation are obscured in knockout mice by the necessity of AKT2 for downstream signalling [22]; however, less complete impairment of AKT activity, such as that observed in diabetic muscle, might have a partial homeostatic effect, by reducing inhibitory feedback via dow…”

“Interestingly, treatment of C2C12 muscle cells with the AMPK agonist 5-aminoimidazole-4carboxamide-1-β-D-ribofuranoside (AICAR) has an additive effect, with insulin, to promote IRS1-associated PI3K activity [145].”

“Similar human studies demonstrate the involvement of mTORC1 in these effects [114], as well as the stimulation by AA of IRS1 S1101 (mS1097) phosphorylation in muscle, likely by S6K1 [56].”

“522–574 of (mouse) IRS1 are required for its ubiquitination and degradation via the cullin 7 E3 ligase [66].”

“In contrast, in Fao or NIH3T3 cells stably expressing IR, overexpression of PKCζ enhances the dissociation from IR and decrease in IRS1 tyrosine phosphorylation caused by prolonged insulin stimulation [95, 96].”

“in all tissues, exhibit abnormal or severely compromised adipocyte differentiation [16, 17].”

“Increased basal phosphorylation of a few IRS S/T—S312 [87], S323 [60], and S636 [91] (mS307, mS318, mS632)—has been described in muscle of type 2 diabetic individuals, whereas basal and stimulated phosphorylation of S330 (mS325) is specifically decreased in muscle of obese insulin-resistant individuals [60].”

“Conversely, increased ERK1/2 phosphorylation of mS612/hS616 is associated with angiotensin II-mediated impairment of endothelial nitric oxide synthase activation (by AKT) in vascular endothelial cells [124].”

“Counter to this idea, the mutation of mS307 to alanine in cultured cells has a rapamycin-like effect, in that it blocks IRS1 disappearance during prolonged insulin stimulation [51, 52]; however, it is not shown that S307A mutation alone alters IRS1 localisation and/or PI3K signalling in these studies.”

“Potentially contributing to the latter finding, ERK1/2 can promote lipolysis and NEFA-mediated insulin resistance, both by downregulating adipocyte perilipin [120] and activating hormone-sensitive lipase [121].”

“In contrast, we used genetic knock-in to replace wild-type IRS1 in mice with a mutant form (S307A, ‘A’) lacking the ability to be phosphorylated at S307 (hS312) [171].”

“Frequent sampling of postprandial blood in these individuals—in combination with more sophisticated tracer/ imaging techniques to monitor metabolism—reveals the redirection of dietary carbohydrate away from glycogen synthesis in muscle and toward the insulin-driven synthesis of TAG in the liver [75, 76].”

“AKT—In contrast to PKCλ, overexpression of membrane-localised AKT (in CHO-T cells) promotes retention of IRS1 tyrosine phosphorylation during prolonged insulin stimulation (after 1 h), with no effect on its acute phosphor-ylation (after 2 min) [104].”

“IRS-deficient muscles also undergo atrophy, with impaired phosphorylation of S6K and its substrate, ribosomal S6 protein [14].”

“signalling is pleiotropic, combined defects in AKT- and aPKC-promoted muscle glucose uptake might be key in the initiation of metabolic disease [23].”

“910–1037) or elsewhere can also impair IRS1–PI3K interaction [63, 64].”

“Yet S302 phosphorylation is implicated in the degradation of IRS1 caused by excessive mTORC1→S6K1 activation [51].”

“Although IR phosphorylates numerous other proteins [9], work with knockout mice has revealed that many insulin responses, including the regulation of carbohydrate and lipid metabolism, require IRS1 and IRS2 [10].”

“The PH domain is the principal module that links IRS1 with the activated IR [26], whereas the PTB domain enhances this interaction via binding to the phospho-NPEY motif within the IR juxtamembrane region [27].”

“Some data suggest that ROCK1 inhibits, rather than promotes, IRS signalling [125], and it has not been demonstrated whether phosphomimetic mutation of mS632 and mS635 can overcome the negative effects of ROCK1 inhibition/knockdown.”

“localisation in insulin-treated cells [57]; however, it remains unclear to what degree the active fraction is S/T phosphorylated.”

“By analogy with JNK1 activity, IRS1 S307 phosphorylation could also have mixed, tissue-specific effects that are obscured by the standard knock-in approach [166].”

“) Serine residues phosphorylated by GSK3 are typically primed by phosphorylation at a nearby site (SXXXS motif), and mutation of both S332 and S336 of IRS1 (hS337, hS341) to alanine enhances both basal and insulin-stimulated tyrosine phosphorylation of IRS1, as well as insulin-stimulated AKT activation in cultured cel…”

“Additionally, AKT S473 phosphorylationmay not fully reflect upstream IRS→PI3K signalling, as non-diabetic but glucose-intolerant individuals can show significantly impaired muscle aPKC activation [83].”

“S789 of IRS1 when co-expressed in cells [143].”

“The tyrosines on IRS1 demonstrated by mutation or MS to recruit dimeric PI3K, GRB2 or SHP2 signalling proteins are indicated by the red bars [18, 178].”

“Some data suggest that ROCK1 inhibits, rather than promotes, IRS signalling [125], and it has not been demonstrated whether phosphomimetic mutation of mS632 and mS635 can overcome the negative effects of ROCK1 inhibition/knockdown.”

“Direct observations of human muscle do not support diminished IRS protein concentration as a primary cause of insulin resistance, though most studies have not examined IRS2 ([80, 81], and reviewed in [4]).”

“mTOR complex 1 (mTORC1) can mediate insulin- or anisomycin-stimulated S307 phosphor-ylation [41], casting some doubt on the significance of insulin-stimulated JNK phosphorylation of S307 observed in cultured cells [42].”

“sensitivity and signalling, improves glucose tolerance, and reduces age-dependent hepatosteatosis [148].”

“skeletal muscle and liver tissues are characterised by increased PKCθ and PKCε levels, respectively [151, 152].”

“impairment of insulin-stimulated AKT activation caused by glucose deprivation [141].”

“PKCδ phosphorylation of S318/ hS323 and other residues may co-opt this chronic function to desensitise insulin signalling [99, 100, 103].”

“Increased plasma concentration of the vasoconstrictive ET-1 polypeptide is associated with insulin resistance and/or hypertension [128, 129], which is, in turn, promoted by direct and indirect (sympathoadrenal and angiotensin II-dependent) effects of compensatory hyperinsulinaemia to cause sodium retention (reviewed i…”

“Though S318A mutation aids retention of IRS1 tyrosine phosphor-ylation during prolonged stimulation, it impairs acute AKT S473 phosphorylation in skeletal muscle cells [101].”

“There may also be a priming effect of S632 (hS636) phosphorylation by rho-associated coiled-coil containing protein kinase 1 (ROCK1) in mice [71, 72].”

“Shulman and colleagues created transgenic mice with moderate overexpression in skeletal muscle (~twofold vs littermates) of nonmutant IRS1, or mutant IRS1 with alanine substitution of three serine residues—S302/307/612A (hS307/312/ 616A)—to block phosphorylation [170].”

“Similarly, mutation of S408 or S357 (hS413, hS362) at least partially blocks the desensitisation of insulin signalling by PKC pathways in PMA-treated cells [157, 158].”

“ET-1 treatment of these cells reduces GLUT4 translocation, both by GRK2 kinase-independent effects on Gαq/11 (or IR), and by decreasing the level and tyrosine phosphorylation of IRS1 [126].”

“Related pathways that contribute to the regulation of AKT include: (1) mTORC1 phosphorylation/ stabilisation of GRB10 [174], which inhibits productive interaction of IRS with a key phosphotyrosine in IR [175]; (2) inhibition of mTORC2 by S6K1-mediated phosphorylation of rictor [176]; and (3) transcriptional upregulati…”

“In this light, it is notable that increased expression of type 1 angiotensin II receptors, promoted by experimental hyperinsulinaemia, correlates with increased IRS1 phosphor-ylation on mS369 [134].”

“) Activation of aPKCs by ceramide can contribute to insulin resistance in mice, though this appears to be due mostly to effects on AKT (reviewed in [94]).”

“In another early study, TNFα—an inflammatory cytokine that can cause insulin resistance in healthy human muscle [34, 35]—was shown to stimulate multi-site S/T phosphorylation of IRS1 and IRS2, blocking their interaction with an IR juxtamembrane domain peptide [36].”

“Diabetic skeletal muscle and liver tissues are characterised by increased PKCθ and PKCε levels, respectively [151, 152].”

“However, mS302 (hS307) phosphorylation in cultured cells potentiates, rather than inhibits, downstream insulin signalling by IRS1 [73].”

“Abbreviations a.a. Amino acid residue AA Complex amino acid mixtures AMPK AMP-activated protein kinase aPKC Atypical protein kinase C cPKC Conventional protein kinase C ERK Extracellular signal-regulated kinase ET-1 Endothelin 1 GPCR G-protein coupled receptor GRB2 Growth factor receptor-bound protein 2 GRK2 G protein…”

“While tissue GRK2 levels have been correlated with plasma norepinephrine/epinephrine levels [132], GRK2 can be upregulated in cultured cells by chronic insulin [89, 90], potentially as a result of PI3K-dependent stabilisation of GRK2 [133].”

“GRK2 can additionally interfere directly with Gαq/11mediated signalling via its RGS (regulator of G protein signalling) domain/GAP activity [127].”

“Interestingly, insulin-stimulated phosphorylation of S307 (mS302) in human primary adipocytes correlates with donor insulin sensitivity [116] and is decreased in adipocytes of humans with type 2 diabetes [117].”

“However, S302, S318 and S570 (hS307, hS323, hS574) are the major sites, and mutation of these residues alleviates inhibition of IRS1 tyrosine phosphorylation by IR in vitro [156].”

“(However, dephosphorylation of basally present phospho-S/Ts is itself inhibitory vs no dephosphorylation [138].”

“aPKC signalling (and/or other steps in the muscle GLUT4 path) might be more sensitive to dysregulation than AKT, as one study found no difference vs control individuals in phosphorylation of AKT (S473) or AKTsubstrate of 160 kDa (AS160), despite the presence of impaired glucose disposal [84].”

“phospho-S612 (hS616) is clearly decreased [122].”

“For example, in 3T3L1 adipocytes, prolonged insulin stimulation causes transit of hyper-S/T-phosphorylated IRS1 from low-density membrane (LDM/high speed pellet) to the cytosol, and this can be inhibited by the mTORC1 inhibitor rapamycin [47, 48].”

“910–1037) or elsewhere can also impair IRS1–PI3K interaction [63, 64].”

“Interestingly, this region of IRS1 contains the human Gly972Arg polymorphism that in some studies is associated with type 2 diabetes risk [61] and might reduce Y941 or Y989 phosphorylation by disrupting an (unknown) IRS1 secondary structure [62].”

“Interestingly, this region of IRS1 contains the human Gly972Arg polymorphism that in some studies is associated with type 2 diabetes risk [61] and might reduce Y941 or Y989 phosphorylation by disrupting an (unknown) IRS1 secondary structure [62].”

“Lee DF, Kuo HP, Chen CT, et al. IKKβ suppression of TSC1 function links the mTOR pathway with insulin resistance.”

“(In human muscle, the PKCι orthologue of PKCλ appears to predominate [93].”

“PKCδ phosphorylation of S318/ hS323 and other residues may co-opt this chronic function to desensitise insulin signalling [99, 100, 103].”

“Conversely, phosphomimetic mutation of S318 to glutamate (S318E) enhances early signalling [70].”

“PKCδ phosphorylation of S318/ hS323 and other residues may co-opt this chronic function to desensitise insulin signalling [99, 100, 103].”

“In 3T3L1 cells, TNFα-stimulated phosphorylation of mS307 is dependent upon the interaction of IRS1 with the NEMO/IKKγ subunit of the IĸB kinase complex, promoted by the actindirected motor protein MYO1C [168].”

“[59]), we emphasise that this regulatory function might be subverted/co-opted by diabetogenic factors—not excluding Copps and White Page 8”

“Regardless, defective muscle IRS1→PI3K→AKT signalling in lean normoglycaemic offspring of type 2 diabetics has been correlated with increased basal S/T phosphorylation of IRS1—including at mTORC1 sites S312 and S636 (mS307, mS632), and S616 (mS612) [85].”

“Of these, phospho-S312, -S616, -S636 and -S1101 are associated with desensitisation of insulin signalling (above and [56, 65]), whereas phospho-S1223 has a sensitising effect [31].”

“S24, altering its membrane-binding properties in cultured cells [29] (Fig.”

“Insulin-stimulated ROCK1 activity is also decreased in muscle of obese diabetic humans, in conjunction with upregulation of its kinase inhibitor RhoE [88].”

“Importantly, and consistent with these data, GRK2-interfering peptides improve insulin sensitivity in diabetic animal models [137]; thus, GRK2 is a potential target for the treatment of insulin resistance.”

“IRS1 phospho-S307 (hS312) increases, contrary to the inhibitory function of S307 phosphorylation in cultured cells [25].”

“Though S318A mutation aids retention of IRS1 tyrosine phosphor-ylation during prolonged stimulation, it impairs acute AKT S473 phosphorylation in skeletal muscle cells [101].”

“Treatment of C2C12 cells with inhibitors implicates JNK and mTORC1 as additional insulin-stimulated S318 kinases [102], demonstrating that multiple pathways may”

“Knockout of the gene encoding PKCβ protects mice against high-fat diet induced obesity, insulin resistance, and hepatosteatosis, but might do so independently of effects on IRS [163].”

“Related pathways that contribute to the regulation of AKT include: (1) mTORC1 phosphor-ylation/ stabilisation of GRB10 [174], which inhibits productive interaction of IRS with a key phosphotyrosine in IR [175]; (2) inhibition of mTORC2 by S6K1-mediated phosphorylation of rictor [176]; and (3) transcriptional upregulat…”

“In contrast, prolonged insulin stimulation in cultured cells can increase PKCα binding to IRS1 in complex with 14-3-3ε [161].”

“Similarly, mutation of S408 or S357 (hS413, hS362) at least partially blocks the desensitisation of insulin signalling by PKC pathways in PMA-treated cells [157, 158].”

“While tissue GRK2 levels have been correlated with plasma norepinephrine/epinephrine levels [132], GRK2 can be upregulated in cultured cells by chronic insulin [89, 90], potentially as a result of PI3Kdependent stabilisation of GRK2 [133].”

“As far less is currently known about IRS2 S/T phosphorylation, we refer the reader to other, limited sources [5, 92].”

“ERK1/2 activation is also sensitised to insulin in adipocytes from humans fed a highenergy diet, potentially contributing to the adaptive expansion of adipose tissue [119].”

“We have shown that insulin-stimulated S522 phosphorylation—which is inhibited by small interfering RNA against AKT—impairs IRS1 tyrosine phosphorylation and PI3K binding in HEK293 cells [58].”

“Rodent and human studies validate the contribution of dyslipidaemia to skeletal muscle and liver insulin resistance via diacylglycerol-mediated activation of nPKCs, including PKCθ [65, 146, 147], PKCδ [148] and PKCε [149, 150] (reviewed in [74, 94]).”

“In FL83B mouse liver cells, glycogen synthesis is upregulated by GRK2 knockdown, and this is associated with decreased basal and insulinstimulated IRS1 S307 (hS312) phosphorylation [136].”

“PKCβ2 can phosphorylate IRS1 on mS336/hS341 in vitro, suggesting it is a priming kinase for GSK3 phosphorylation of IRS1 [162].”

“However, PKCζ can phosphorylate IRS1 in vitro on S498, S570 and S612 (hS503, hS574, hS616), with phospho-S570 having an adverse effect on PI3K binding [98].”

“conjunction with increased AKT activation [123].”

“PKCα in rodent muscle associates basally with IRS1; insulin transiently disrupts this interaction while increasing PKCα association with AKT [160].”

“Potentially then, the disruptive effect of S307 phosphorylation might be an assay- or condition-dependent aspect of a more generalised S/T phosphorylation-based mechanism governing IRS signalling [5, 45, 46].”

“Compatible with regulated ubiquitin/proteasomal processing of IRS1, exposure of HEK293 cells to very high insulin showed that about 50% of total IRS1 could be degraded within 10 min via internal cleavage around hS475 or hS641; however, the necessary factors were not investigated [67].”

“IRS1 tyrosine phosphorylation/PI3K activity or glucose uptake [169].”

“This process creates a suitable binding site for IRS‐1 that is then activated via phosphorylation by different insulin‐induced kinases such as PKC, SIK2, AKT, S6K1, mTOR, extracellular signal‐regulated kinase 1/2 (ERK1/2), and ROCK1 (Copps & White, 2012; Kiselyov et al., 2009).”

“A plethora of evidence suggests AGEs/RAGE signaling pathway, NFκB activation, inflammation, and ROS generation are directly related to the pathogenesis of insulin resistance by increased IRS-1serine phosphorylation and degradation, thus blocking the insulin signaling pathway [68,71,73,74].”

“Several types of insulin-dependent kinases such as ERK1/2 (extracellular signal-regulated kinase 1/2), atypical PKC (protein kinase C), S6K1 (ribosomal protein S6 kinase beta-1), SIK2 (serine/threonine-protein kinase 2), Akt (protein kinase B), mTOR (mammalian target of rapamycin), and ROCK1 (rho-associated protein ki…”

“In addition, mTORC1 signaling can negatively regulate RTK signaling to reduce both PI3K-AKT-mTOR and RAS/MAPK activity, including mTORC1-S6K-mediated suppression of the insulin receptor substrate protein IRS1; a major IGF-1 receptor substrate and adaptor protein that can promote both PI3K and RAS activation by binding…”

“IRS proteins are characterized by the presence of a NH2-terminal pleckstrin homology (PH) domain adjacent to a phosphotyrosine-binding (PTB) domain, followed by a COOH-terminal tail that contains numerous tyrosine and serine/threonine phosphorylation sites (Copps & White 2012).”

“residues phosphorylation in the IRS and desensitization of proximal insulin signaling plays an important role in the pathogenesis of IR (127).”

“Mechanistically, HCV core protein leads to upregulation of inflammatory markers such as tumor necrosis factor α (TNF-α), eventually resulting in reduced downstream activation of insulin signaling [36].”

“As only phospho-Ser/Thr on IRS-1 can have negative or positive effects on the insulin signaling of healthy and diseased tissues, those molecules might play an important pathophysiological role (292).”

“…feedback within individual systems, these events seem likely to act in cell types under different conditions (Carracedo and Pandolfi, 2008; Copps and White, 2012 One simple direct analysis of feedback between GSK3 and AKT was by a human kinome siRNA knockdown screen looking for kinases…”

“…autophosphorylation by inducing structural rearrangement of the transmembrane domains to bring them into close proximity with each other (Gutmann et al., 2018), and the consequent activation of the IR tyrosine kinase toward phosphorylation of its major substrates IRS1,2 (Copps and White, 2012).”

“When present in excess, several of these lipid intermediates have been suggested to induce insulin resistance in ectopic tissues by interfering with insulin signaling at the level of the insulin receptor substrate (IRS) proteins (Boura-Halfon & Zick 2009, Copps & White 2012).”

“These proteins are tightly regulated by phosphorylation on both positive tyrosine and inhibitory serine/threonine residues [24].”

“JNK changes the function of insulin receptor substrate 1 (IRS-1), which regulates insulin and insulin-like growth factor pathways.(45) These pathways play an important role in age-related disease and life span in animal models.”

“Serine/threonine phosphorylation of IRS affects its tyrosine dephosphorylation, its dissociation from IR, its intracellular localization, and its eventual degradation in a proteasome dependent manner through the different mechanisms [58,75,76].”

“TNFα inhibit insulin signaling by activation of the JNK 1/2 signaling pathway which phosphorylates insulin receptor substrate 1 (70).”

“Although many genetic and physiological factors interact to produce and aggravate insulin resistance, a common underlying mechanism is dysregulated insulin signaling by insulin receptor substrates (IRS1 and IRS2) that are inappropriately phosphorylated on serine residues (1).”

“IRS-1 serine 307 mutated to alanine had reduced insulin sensitivity (Copps et al., 2010; Copps and White, 2012).”

“In physiological condition, insulin induces a time-controlled phosphorylation of both positive and inhibitory serine sites in IRS1 and IRS2 in order to ensure the fine tuning of IRS tyrosine phosphorylation that is necessary for the propagation of insulin action (Tanti and Jager, 2009; Copps and White, 2012).”

“ERK proteins are one of the best-characterized groups of proteins able to phosphorylate IRSs [35,36].”

“The phosphorylated receptor then activates insulin receptor substrate (IRS) proteins at Tyr residue, which subsequently binds to the SH2 domain of the regulatory subunit of PI3K leading to its departure from the catalytic subunit (Copps and White 2012).”

“…for establishing pathogenic feed-forward loops that inhibit normal insulin signaling, as mTORc, ribosomal protein S6 kinase beta-1 (S6K1), and GSK3-β induce hyperphosphorylation at various Ser residues (S632, S302/S522 and S337, respectively; EldarFinkelman and Krebs, 1997; Copps and White, 2012).”

“IRS proteins also contain >50 serine/ threonine phosphorylation sites (shown as red diamonds in Figure 2), which modulate stability and tyrosine phosphorylation for feedback and heterologous regulation [23].”

“In muscle of obese diabetic human subjects, insulin-stimulated ROCK1 was noticed to be decreased with up-regulation of its kinase inhibitor RhoE [144].”

“The tail of IRS1 contains distinct tyrosine and serine phosphorylation sites, which serve as the docking locus for SH2 homology signal transducers [20,46].”

“…via the phosphorylation of several key serine residues (such as mouse S265/S302/S522/S1097, human S270/S307/ S527/S1101 by S6K1, mouse S307/S612/S632, and human S312/S616/S636 by mTORC1) found by in vitro kinase assays and/or mutation studies in cells and mice (reviewed in Copps and White, 2012).”

“Most of the insulin-mediated metabolic actions are controlled by the PI3K–AKT/PKB pathway, which is phosphorylated by the insulin receptor through two major substrates, IRS-1 and IRS-2 [34].”

“IRS1 and IRS2 serine or threonine phosphorylation is believed to mediate IRS degradation, ubiquitination, and insulin resistance (25).”

“Unlike Tyr phosphorylation by the IR, which activates IRS, modifications at Ser/Thr residues are capable of both promoting and inhibiting downstream IRS-mediated signaling [57].”

“However, phosphorylation of residues adjacent to the PI3K binding sites by serine kinases displaces PI3K and thus interferes with its ability to activate IRS1.(90,94) The inhibitory effect of free FAs on IRS1 and insulin signaling was demonstrated in cell culture,(95,96) animal models, and human volunteers(100) and ma…”

“The downstream events that follow INSR activation include recruitment of several adaptor proteins, facilitating a suitable binding site for insulin receptor substrates (IRSs) [82] that once phosphorylated, trigger several downstream signals [83].”

“A sort of feedback of serine/threonine phosphorylation of IRS underlies the magnitude of insulin stimulation (34).”

“Mechanistically, since increased p85α expression and IRS1 serine phosphorylation are essential for the induction of clinically-apparent IR [32], we demonstrated significant increase in the p-IRS1/IRS1 ratio (123%, p < 0.”

“Specific changes in the phosphorylation pattern of IRS-1 resulting in impaired insulin signaling have long been established as pathological markers of IR in peripheral tissues [Copps and White, 2012; Hoehn et al., 2008; Taniguchi et al., 2006].”

“Indeed pro-inflammatory cytokines can activate downstream kinases of insulin signaling pathway, including IκB kinase-β (IKKβ), JUN amino-terminal kinase 1 (JNK1) and p38 MAPK, which can contribute to increased serine phosphorylation that inactivates IRS protein and stimulate the production of suppressors of cytokine s…”

“In turn, proinflammatory cytokine release mediated by immune cells induces serine phosphorylation of insulin receptor substrate-1 (IRS-1) leading to local and systemic insulin resistance, which is thought to contribute toward whole body glucose and fatty acid metabolic dysregulation (24, 25) Furthermore, the obese adi…”

“JNK promotes the serine phosphorylation of IRS-1 that inhibits insulin-dependent IRS-1 tyrosine phosphorylation and downstream signaling (85, 86).”

“114,169 Mechanically, the TLR4-mediated activation of the IKK β and JNK pathways, likely via MYD88, is thought to directly mediate FA-induced insulin resistance 170 by inducing the serine/ threonine phosphorylation of IRSs and reducing their responsiveness to insulin.”

“The literature is replete with data implicating the phosphorylation of IRS1 at different serine/threonine residues in positive or negative control of insulin signaling and in positive or negative feedback loops of insulin signaling (16).”

“, PKC, AKT, mammalian target of rapamycin (mTOR), and extracellular regulated protein kinase (ERK)1/2) [38,39].”

“(n = 3, mean ± SEM, one-sample t test, *p < 0.01). negative feedback loop that triggers insulin resistance and increased adiposity (Copps and White, 2012; Hsu et al., 2011; Um et al., 2004; Yu et al., 2011).”

“Insulin resistance in adipocytes is associated with impaired phosphorylation of insulin receptor substrate 1 (IRS-1) in obesity, which is further impaired in T2DM (Copps and White, 2012).”

“With respect to insulin-dependent signalling pathway, IRS-1 phosphorylation is altered in patients with diabetes.([16]) AMPK (cellular energy sensor) and its upstream kinase (LKB1) can be activated under ATP-depleted metabolic states, and these pathways are essential in promotion of glucose uptake and fat oxidation.”

“Another putative target, insulin receptor substrate-2 (IRS2) , was confi rmed as miR-33 target by Fernandez-Hernando’s group [ 19 ]—IRS-2, together with IRS-1, are both important for transduction of insulin signaling over the cell membrane and their blockade thus interferes with insulin effects on the cells [ 71 ].”

“Dysregulated serine phosphorylation of IRS-1 and IRS-2 represents a key mechanism to promote insulin resistance by interference of IRS-1/2 tyrosine phosphorylation and/or increased proteolytic degradation (Copps and White, 2012).”

“At baseline, obese subjects had increased phosphorylation and thus activation of JNK and pIKK-β with increased inhibitory serine phosphorylation of IRS-1 and reduced GLUT4 response to insulin.”

“Because a limited number of phospho-specific antibodies were generated in early studies, the phosphorylation of a few Ser/Thr residues (Ser302, Ser-307, Ser-318, Ser-612, and Ser-632) has been interrogated in many different contexts, whereas many others have never been examined (6).”

“Both metabolic and mechanic stresses activate intracellular protein kinases that trigger serine or threonine phosphorylation of IRS proteins, which couple with IRS ubiquitination and degradation (Copps & White 2012).”

“Retinal Müller cells: a primary site of IRS signalling in the retina IRS proteins are heavily phosphorylated on tyrosine, serine and threonine residues [37, 47, 48].”

“(2) The insulin signal-transduction efficacy relies strongly on the phosphorylation of IRS1 at different sites (Ser/Tyr), which is correlated with the subcellular relocalization and proteasome-mediated degradation of IRS1.”

“Insulin signaling is negatively regulated by a number of mechanisms, such as the binding of the adaptor protein Grb10 to the kinase domain of IR ((8), (9)), serine phosphorylation of IRS1 ((10), (11)), and dephosphorylation of IR and IRS1/2 by protein tyrosine phosphatase-1B (PTP-1B) (12).”

“IRS-1 can be also activated by other kinases including ERK1/25, atypical PKC6, S6K17, SIK28, AKT, mTOR9 and ROCK110 as well as other types of kinases such as AMPK11 and GSK312 [33, 34].”

“Indeed, once persistently stimulated, mTOR is able to phosphorylate IRS1 on Ser residues critical for BIR, including Ser307 [19,42] and previous studies from both our groups [43,44] and others [45–48] reported about the hyper-activation of mTOR in AD.”