Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

The insulin signalling network

Nature Metabolism volume 7, pages 1745–1764 (2025)Cite this article

Subjects

Abstract

Insulin signalling is a central regulator of metabolism, orchestrating nutrient homeostasis and coordinating carbohydrate, protein and lipid metabolism. This network operates through dynamic, tightly regulated protein phosphorylation events involving key kinases such as AKT, shaping cellular responses with remarkable precision. Advances in phosphoproteomics have expanded our understanding of insulin signalling, revealing its intricate regulation and links to disease, particularly cardiometabolic disease. Major insights, such as the mechanisms of AKT activation and the influence of genetic and environmental factors, have emerged from studying this network. In this Review, we examine the architecture of insulin signalling, focusing on its precise temporal regulation. We highlight AKT’s central role in insulin action and its vast substrate repertoire, which governs diverse cellular functions. Additionally, we explore feedback and crosstalk mechanisms, such as insulin receptor substrate protein signalling, which integrates inputs through phosphorylation at hundreds of distinct sites. Crucially, phosphoproteomics has uncovered complexities in insulin-resistant states, where network rewiring is characterized by disrupted phosphorylation and the emergence of novel sites that are absent in healthy cells. These insights redefine insulin signalling and its dysfunction, highlighting new therapeutic opportunities.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Timeline of breakthroughs in the insulin pathway.
Fig. 2: Canonical insulin signalling pathway.
Fig. 3: Phosphorylation sites in IRS proteins.
Fig. 4: Feedback mechanisms within the ISN.
Fig. 5: Insulin signalling rewiring in insulin resistance.

Similar content being viewed by others

References

  1. Krebs, E. G. & Fischer, E. H. Phosphorylase activity of skeletal muscle extracts. J. Biol. Chem. 216, 113–120 (1955).

    Article  PubMed  CAS  Google Scholar 

  2. Fischer, E. H. & Krebs, E. G. Conversion of phosphorylase b to phosphorylase a in muscle extracts. J. Biol. Chem. 216, 121–132 (1955).

    Article  PubMed  CAS  Google Scholar 

  3. Kresge, N., Simoni, R. D. & Hill, R. L. The process of reversible phosphorylation: the work of Edmond H. Fischer. J. Biol. Chem. 286, e1–e2 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Nimmo, H. G. & Cohen, P. Glycogen synthetase kinase 2 (GSK 2); the identification of a new protein kinase in skeletal muscle. FEBS Lett. 47, 162–166 (1974).

    Article  PubMed  CAS  Google Scholar 

  5. Jungas, R. L. Hormonal regulation of pyruvate dehydrogenase. Metabolism 20, 43–53 (1971).

    Article  PubMed  CAS  Google Scholar 

  6. Riley, N. M. & Coon, J. J. Phosphoproteomics in the age of rapid and deep proteome profiling. Anal. Chem. 88, 74–94 (2016).

    Article  PubMed  CAS  Google Scholar 

  7. Kearney, A. L. et al. Akt phosphorylates insulin receptor substrate to limit PI3K-mediated PIP3 synthesis. eLife 10, e66942 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Norris, D. M. et al. An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction. J. Cell Sci. 130, 2757–2766 (2017).

    Article  PubMed  CAS  Google Scholar 

  9. Lee, S. & Dong, H. H. FoxO integration of insulin signaling with glucose and lipid metabolism. J. Endocrinol. 233, R67–R79 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Humphrey, S. J. et al. Dynamic adipocyte phosphoproteome reveals that Akt directly regulates mTORC2. Cell Metab. 17, 1009–1020 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Humphrey, S. J., Azimifar, S. B. & Mann, M. High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics. Nat. Biotechnol. 33, 990–995 (2015).

    Article  PubMed  CAS  Google Scholar 

  12. Choi, E. et al. Mitotic regulators and the SHP2–MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling. Nat. Commun. 10, 1473 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Cai, W. et al. Domain-dependent effects of insulin and IGF-1 receptors on signalling and gene expression. Nat. Commun. 8, 14892 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Svendsen, A. M. et al. Down-regulation of cyclin G2 by insulin, IGF-I (insulin-like growth factor 1) and X10 (AspB10 insulin): role in mitogenesis. Biochem. J. 457, 69–77 (2014).

    Article  PubMed  CAS  Google Scholar 

  15. Zhou, H.-L. et al. An enzyme that selectively S-nitrosylates proteins to regulate insulin signaling. Cell 186, 5812–5825 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Chen, C. et al. Cullin neddylation inhibitor attenuates hyperglycemia by enhancing hepatic insulin signaling through insulin receptor substrate stabilization. Proc. Natl Acad. Sci. USA 119, e2111737119 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Su, Z. et al. Global redox proteome and phosphoproteome analysis reveals redox switch in Akt. Nat. Commun. 10, 5486 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Haeusler, R. A., McGraw, T. E. & Accili, D. Biochemical and cellular properties of insulin receptor signalling. Nat. Rev. Mol. Cell Biol. 19, 31–44 (2018).

    Article  PubMed  CAS  Google Scholar 

  19. van Gerwen, J., Shun-Shion, A. S. & Fazakerley, D. J. Insulin signalling and GLUT4 trafficking in insulin resistance. Biochem. Soc. Trans. 51, 1057–1069 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Saltiel, A. R. Insulin signaling in health and disease. J. Clin. Invest. 131, e142241 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. James, D. E., Stöckli, J. & Birnbaum, M. J. The aetiology and molecular landscape of insulin resistance. Nat. Rev. Mol. Cell Biol. 22, 751–771 (2021).

    Article  PubMed  CAS  Google Scholar 

  22. Pørksen, N. et al. Pulsatile insulin secretion: detection, regulation, and role in diabetes. Diabetes 51, S245–S254 (2002).

    Article  PubMed  Google Scholar 

  23. White, M. F. & Kahn, C. R. Insulin action at a molecular level—100 years of progress. Mol. Metab. 52, 101304 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Choi, E. & Bai, X.-C. The activation mechanism of the insulin receptor: a structural perspective. Annu. Rev. Biochem. 92, 247–272 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Liao, Z. et al. Comprehensive insulin receptor phosphorylation dynamics profiled by mass spectrometry. FEBS J. 289, 2657–2671 (2022).

    Article  PubMed  CAS  Google Scholar 

  26. Myers, M. G. Jr. & White, M. F. Insulin signal transduction and the IRS proteins. Annu. Rev. Pharmacol. Toxicol. 36, 615–658 (1996).

    Article  PubMed  CAS  Google Scholar 

  27. Hanke, S. & Mann, M. The phosphotyrosine interactome of the insulin receptor family and its substrates IRS-1 and IRS-2. Mol. Cell. Proteomics 8, 519–534 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Alberti, S. & Hyman, A. A. Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing. Nat. Rev. Mol. Cell Biol. 22, 196–213 (2021).

    Article  PubMed  CAS  Google Scholar 

  29. Melvin, A., O’Rahilly, S. & Savage, D. B. Genetic syndromes of severe insulin resistance. Curr. Opin. Genet. Dev. 50, 60–67 (2018).

    Article  PubMed  CAS  Google Scholar 

  30. Nagao, H. et al. Unique ligand and kinase-independent roles of the insulin receptor in regulation of cell cycle, senescence and apoptosis. Nat. Commun. 14, 57 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Haeusler, R. A. & Accili, D. The double life of Irs. Cell Metab. 8, 7–9 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Tseng, Y.-H., Kriauciunas, K. M., Kokkotou, E. & Kahn, C. R. Differential roles of insulin receptor substrates in brown adipocyte differentiation. Mol. Cell. Biol. 24, 1918–1929 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Toyoshima, Y. et al. Deletion of IRS-1 leads to growth failure and insulin resistance with downregulation of liver and muscle insulin signaling in rats. Sci. Rep. 15, 649 (2025).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Tamemoto, H. et al. Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature 372, 182–186 (1994).

    Article  PubMed  CAS  Google Scholar 

  35. Araki, E. et al. Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372, 186–190 (1994).

    Article  PubMed  CAS  Google Scholar 

  36. Kubota, N. et al. Disruption of insulin receptor substrate 2 causes type 2 diabetes because of liver insulin resistance and lack of compensatory beta-cell hyperplasia. Diabetes 49, 1880–1889 (2000).

    Article  PubMed  CAS  Google Scholar 

  37. Toyoshima, Y. et al. Disruption of insulin receptor substrate-2 impairs growth but not insulin function in rats. J. Biol. Chem. 295, 11914–11927 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Nolte, R. T., Eck, M. J., Schlessinger, J., Shoelson, S. E. & Harrison, S. C. Crystal structure of the PI 3-kinase p85 amino-terminal SH2 domain and its phosphopeptide complexes. Nat. Struct. Biol. 3, 364–374 (1996).

    Article  PubMed  CAS  Google Scholar 

  39. Madsen, R. R. & Toker, A. PI3K signaling through a biochemical systems lens. J. Biol. Chem. 299, 105224 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Luo, J., Field, S. J., Lee, J. Y., Engelman, J. A. & Cantley, L. C. The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex. J. Cell Biol. 170, 455–464 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Myers, M. G. Jr. et al. IRS-1 activates phosphatidylinositol 3′-kinase by associating with src homology 2 domains of p85. Proc. Natl Acad. Sci. USA 89, 10350–10354 (1992).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Liu, G. Y. & Sabatini, D. M. mTOR at the nexus of nutrition, growth, ageing and disease. Nat. Rev. Mol. Cell Biol. 21, 183–203 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Ebner, M., Sinkovics, B., Szczygieł, M., Ribeiro, D. W. & Yudushkin, I. Localization of mTORC2 activity inside cells. J. Cell Biol. 216, 343–353 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Madsen, R. R. & Vanhaesebroeck, B. Cracking the context-specific PI3K signaling code. Sci. Signal. 13, eaay2940 (2020).

    Article  PubMed  CAS  Google Scholar 

  45. Manning, B. D. & Toker, A. AKT/PKB signaling: navigating the network. Cell 169, 381–405 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Hill, M. M. et al. A role for protein kinase Bβ/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes. Mol. Cell. Biol. 19, 7771–7781 (1999).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Gonzalez, E. & McGraw, T. E. The Akt kinases: isoform specificity in metabolism and cancer. Cell Cycle 8, 2502–2508 (2009).

    Article  PubMed  CAS  Google Scholar 

  48. Sanidas, I. et al. Phosphoproteomics screen reveals Akt isoform-specific signals linking RNA processing to lung cancer. Mol. Cell 53, 577–590 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Reinartz, M., Raupach, A., Kaisers, W. & Gödecke, A. AKT1 and AKT2 induce distinct phosphorylation patterns in HL-1 cardiac myocytes. J. Proteome Res. 13, 4232–4245 (2014).

    Article  PubMed  CAS  Google Scholar 

  50. Manning, B. D. & Cantley, L. C. AKT/PKB signaling: navigating downstream. Cell 129, 1261–1274 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Liu, P. et al. PtdIns(3,4,5)P3-dependent activation of the mTORC2 kinase complex. Cancer Discov. 5, 1194–1209 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Calleja, V. et al. Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo. PLoS Biol. 5, e95 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Kearney, A. L. et al. Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes. J. Biol. Chem. 294, 16729–16739 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Bozulic, L., Surucu, B., Hynx, D. & Hemmings, B. A. PKBα/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. Mol. Cell 30, 203–213 (2008).

    Article  PubMed  CAS  Google Scholar 

  55. Mora, A., Komander, D., van Aalten, D. M. F. & Alessi, D. R. PDK1, the master regulator of AGC kinase signal transduction. Semin. Cell Dev. Biol. 15, 161–170 (2004).

    Article  PubMed  CAS  Google Scholar 

  56. Collins, B. J., Deak, M., Arthur, J. S. C., Armit, L. J. & Alessi, D. R. In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation. EMBO J. 22, 4202–4211 (2003).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. McManus, E. J. et al. The in vivo role of PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation. EMBO J. 23, 2071–2082 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Guo, H. et al. Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules. Oncogene 33, 3463–3472 (2014).

    Article  PubMed  CAS  Google Scholar 

  59. Di Maira, G. et al. Protein kinase CK2 phosphorylates and upregulates Akt/PKB. Cell Death Differ. 12, 668–677 (2005).

    Article  PubMed  Google Scholar 

  60. Menon, S. et al. Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell 156, 771–785 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Tee, A. R., Manning, B. D., Roux, P. P., Cantley, L. C. & Blenis, J. Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Curr. Biol. 13, 1259–1268 (2003); erratum 32, 733–734 (2022).

    Article  PubMed  CAS  Google Scholar 

  62. Inoki, K., Li, Y., Xu, T. & Guan, K.-L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 17, 1829–1834 (2003).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Vander Haar, E., Lee, S.-I., Bandhakavi, S., Griffin, T. J. & Kim, D.-H. Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat. Cell Biol. 9, 316–323 (2007).

    Article  Google Scholar 

  64. Sancak, Y. et al. PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol. Cell 25, 903–915 (2007).

    Article  PubMed  CAS  Google Scholar 

  65. Laplante, M. & Sabatini, D. M. mTOR signaling in growth control and disease. Cell 149, 274–293 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  66. Sancak, Y. et al. Ragulator–Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290–303 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Fernandes, S. A. et al. Spatial and functional separation of mTORC1 signalling in response to different amino acid sources. Nat. Cell Biol. 26, 1918–1933 (2024).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  68. Zhou, X. et al. Dynamic visualization of mTORC1 activity in living cells. Cell Rep. 10, 1767–1777 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  69. Shin, H. R. et al. Lysosomal GPCR-like protein LYCHOS signals cholesterol sufficiency to mTORC1. Science 377, 1290–1298 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. Koundouros, N. et al. Direct sensing of dietary ω-6 linoleic acid through FABP5–mTORC1 signaling. Science 387, eadm9805 (2025).

    Article  PubMed  CAS  Google Scholar 

  71. Frias, M. A. et al. Phosphatidic acid drives mTORC1 lysosomal translocation in the absence of amino acids. J. Biol. Chem. 295, 263–274 (2020).

    Article  PubMed  CAS  Google Scholar 

  72. Bodine, S. C. et al. Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat. Cell Biol. 3, 1014–1019 (2001).

    Article  PubMed  CAS  Google Scholar 

  73. Porstmann, T. et al. SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth. Cell Metab. 8, 224–236 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  74. Düvel, K. et al. Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol. Cell 39, 171–183 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  75. Lee, P. L., Jung, S. M. & Guertin, D. A. The complex roles of mechanistic target of rapamycin in adipocytes and beyond. Trends Endocrinol. Metab. 28, 319–339 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  76. Orozco, J. M. et al. Dihydroxyacetone phosphate signals glucose availability to mTORC1. Nat. Metab. 2, 893–901 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  77. Suzawa, M. & Bland, M. L. Insulin signaling in development. Development 150, dev201599 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  78. Fingar, D. C. & Birnbaum, M. J. Characterization of the mitogen-activated protein kinase/90-kilodalton ribosomal protein S6 kinase signaling pathway in 3T3-L1 adipocytes and its role in insulin-stimulated glucose transport. Endocrinology 134, 728–735 (1994).

    Article  PubMed  CAS  Google Scholar 

  79. Ng, Y., Ramm, G. & James, D. E. Dissecting the mechanism of insulin resistance using a novel heterodimerization strategy to activate Akt. J. Biol. Chem. 285, 5232–5239 (2010).

    Article  PubMed  CAS  Google Scholar 

  80. Lavoie, H., Gagnon, J. & Therrien, M. ERK signalling: a master regulator of cell behaviour, life and fate. Nat. Rev. Mol. Cell Biol. 21, 607–632 (2020).

    Article  PubMed  CAS  Google Scholar 

  81. Yamauchi, K. & Pessin, J. E. Insulin receptor substrate-1 (IRS1) and Shc compete for a limited pool of Grb2 in mediating insulin downstream signaling. J. Biol. Chem. 269, 31107–31114 (1994).

    Article  PubMed  CAS  Google Scholar 

  82. Faisal, A., el-Shemerly, M., Hess, D. & Nagamine, Y. Serine/threonine phosphorylation of ShcA. Regulation of protein-tyrosine phosphatase–PEST binding and involvement in insulin signaling. J. Biol. Chem. 277, 30144–30152 (2002).

    Article  PubMed  CAS  Google Scholar 

  83. Hanafusa, H., Torii, S., Yasunaga, T., Matsumoto, K. & Nishida, E. Shp2, an SH2-containing protein-tyrosine phosphatase, positively regulates receptor tyrosine kinase signaling by dephosphorylating and inactivating the inhibitor Sprouty. J. Biol. Chem. 279, 22992–22995 (2004).

    Article  PubMed  CAS  Google Scholar 

  84. Uehara, T., Tokumitsu, Y. & Nomura, Y. Wortmannin inhibits insulin-induced Ras and mitogen-activated protein kinase activation related to adipocyte differentiation in 3T3-L1 fibroblasts. Biochem. Biophys. Res. Commun. 210, 574–580 (1995).

    Article  PubMed  CAS  Google Scholar 

  85. Liu, L., Xie, Y. & Lou, L. PI3K is required for insulin-stimulated but not EGF-stimulated ERK1/2 activation. Eur. J. Cell Biol. 85, 367–374 (2006).

    Article  PubMed  CAS  Google Scholar 

  86. Bard-Chapeau, E. A. et al. Deletion of Gab1 in the liver leads to enhanced glucose tolerance and improved hepatic insulin action. Nat. Med. 11, 567–571 (2005).

    Article  PubMed  CAS  Google Scholar 

  87. Gu, H. & Neel, B. G. The “Gab” in signal transduction. Trends Cell Biol. 13, 122–130 (2003).

    Article  PubMed  CAS  Google Scholar 

  88. Lopez, J. A. et al. Identification of a distal GLUT4 trafficking event controlled by actin polymerization. Mol. Biol. Cell 20, 3918–3929 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  89. Nishita, M. et al. Phosphoinositide 3-kinase-mediated activation of cofilin phosphatase Slingshot and its role for insulin-induced membrane protrusion. J. Biol. Chem. 279, 7193–7198 (2004).

    Article  PubMed  CAS  Google Scholar 

  90. Chiu, T. T., Patel, N., Shaw, A. E., Bamburg, J. R. & Klip, A. Arp2/3- and cofilin-coordinated actin dynamics is required for insulin-mediated GLUT4 translocation to the surface of muscle cells. Mol. Biol. Cell 21, 3529–3539 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  91. Masson, S. W. C., Sorrenson, B., Shepherd, P. R. & Merry, T. L. β-catenin regulates muscle glucose transport via actin remodelling and M-cadherin binding. Mol. Metab. 42, 101091 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  92. Zhou, G.-L. et al. Akt phosphorylation of serine 21 on Pak1 modulates Nck binding and cell migration. Mol. Cell. Biol. 23, 8058–8069 (2003).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  93. Møller, L. L. V. et al. Insulin-stimulated glucose uptake partly relies on p21-activated kinase (PAK)2, but not PAK1, in mouse skeletal muscle. J. Physiol. 598, 5351–5377 (2020).

    Article  PubMed  Google Scholar 

  94. Ueda, S. et al. Crucial role of the small GTPase Rac1 in insulin-stimulated translocation of glucose transporter 4 to the mouse skeletal muscle sarcolemma. FASEB J. 24, 2254–2261 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  95. Sylow, L. et al. Rac1 signaling is required for insulin-stimulated glucose uptake and is dysregulated in insulin-resistant murine and human skeletal muscle. Diabetes 62, 1865–1875 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  96. Ishikura, S., Koshkina, A. & Klip, A. Small G proteins in insulin action: Rab and Rho families at the crossroads of signal transduction and GLUT4 vesicle traffic. Acta Physiol. (Oxf.) 192, 61–74 (2008).

    Article  PubMed  CAS  Google Scholar 

  97. Hodge, R. G. & Ridley, A. J. Regulating Rho GTPases and their regulators. Nat. Rev. Mol. Cell Biol. 17, 496–510 (2016).

    Article  PubMed  CAS  Google Scholar 

  98. Payapilly, A. & Malliri, A. Compartmentalisation of RAC1 signalling. Curr. Opin. Cell Biol. 54, 50–56 (2018).

    Article  PubMed  CAS  Google Scholar 

  99. Hu, H. et al. Phosphoinositide 3-kinase regulates glycolysis through mobilization of aldolase from the actin cytoskeleton. Cell 164, 433–446 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  100. Sylow, L. et al. Akt and Rac1 signaling are jointly required for insulin-stimulated glucose uptake in skeletal muscle and downregulated in insulin resistance. Cell. Signal. 26, 323–331 (2014).

    Article  PubMed  CAS  Google Scholar 

  101. Møller, L. L. V. et al. The Rho guanine dissociation inhibitor α inhibits skeletal muscle Rac1 activity and insulin action. Proc. Natl Acad. Sci. USA 120, e2211041120 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  102. Chiang, S. H. et al. Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10. Nature 410, 944–948 (2001).

    Article  PubMed  CAS  Google Scholar 

  103. Wang, S. et al. Inducible Exoc7/Exo70 knockout reveals a critical role of the exocyst in insulin-regulated GLUT4 exocytosis. J. Biol. Chem. 294, 19988–19996 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  104. Liu, J., Kimura, A., Baumann, C. A. & Saltiel, A. R. APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3-L1 adipocytes. Mol. Cell. Biol. 22, 3599–3609 (2002).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  105. Hu, J., Liu, J., Ghirlando, R., Saltiel, A. R. & Hubbard, S. R. Structural basis for recruitment of the adaptor protein APS to the activated insulin receptor. Mol. Cell 12, 1379–1389 (2003).

    Article  PubMed  CAS  Google Scholar 

  106. Ahn, M.-Y., Katsanakis, K. D., Bheda, F. & Pillay, T. S. Primary and essential role of the adaptor protein APS for recruitment of both c-Cbl and its associated protein CAP in insulin signaling. J. Biol. Chem. 279, 21526–21532 (2004).

    Article  PubMed  CAS  Google Scholar 

  107. Chang, T.-J. et al. Genetic variation of SORBS1 gene is associated with glucose homeostasis and age at onset of diabetes: a SAPPHIRe Cohort Study. Sci. Rep. 8, 10574 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  108. Gong, S. et al. A variation in SORBS1 is associated with type 2 diabetes and high-density lipoprotein cholesterol in Chinese population. Diabetes Metab. Res. Rev. 38, e3524 (2022).

    Article  PubMed  CAS  Google Scholar 

  109. Ng, Y., Ramm, G., Lopez, J. A. & James, D. E. Rapid activation of Akt2 is sufficient to stimulate GLUT4 translocation in 3T3-L1 adipocytes. Cell Metab. 7, 348–356 (2008).

    Article  PubMed  CAS  Google Scholar 

  110. Kohn, A. D., Summers, S. A., Birnbaum, M. J. & Roth, R. A. Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J. Biol. Chem. 271, 31372–31378 (1996).

    Article  PubMed  CAS  Google Scholar 

  111. Katsura, Y. et al. An optogenetic system for interrogating the temporal dynamics of Akt. Sci. Rep. 5, 14589 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  112. Moritz, A. et al. Akt–RSK–S6 kinase signaling networks activated by oncogenic receptor tyrosine kinases. Sci. Signal. 3, ra64 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  113. Horman, S. et al. Insulin antagonizes ischemia-induced Thr172 phosphorylation of AMP-activated protein kinase α-subunits in heart via hierarchical phosphorylation of Ser485/491. J. Biol. Chem. 281, 5335–5340 (2006).

    Article  PubMed  CAS  Google Scholar 

  114. Berwick, D. C., Hers, I., Heesom, K. J., Moule, S. K. & Tavareá, J. M. The identification of ATP-citrate lyase as a protein kinase B (Akt) substrate in primary adipocytes. J. Biol. Chem. 277, 33895–33900 (2002).

    Article  PubMed  CAS  Google Scholar 

  115. Luo, M. et al. Phosphorylation of human insulin receptor substrate-1 at serine 629 plays a positive role in insulin signaling. Endocrinology 148, 4895–4905 (2007).

    Article  PubMed  CAS  Google Scholar 

  116. Sano, H. et al. Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation. J. Biol. Chem. 278, 14599–14602 (2003).

    Article  PubMed  CAS  Google Scholar 

  117. Brunet, A. et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868 (1999).

    Article  PubMed  CAS  Google Scholar 

  118. Stöckli, J. et al. ABHD15 regulates adipose tissue lipolysis and hepatic lipid accumulation. Mol. Metab. 25, 83–94 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  119. Inoki, K., Li, Y., Zhu, T., Wu, J. & Guan, K.-L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4, 648–657 (2002).

    Article  PubMed  CAS  Google Scholar 

  120. Pennington, K. L., Chan, T. Y., Torres, M. P. & Andersen, J. L. The dynamic and stress-adaptive signaling hub of 14-3-3: emerging mechanisms of regulation and context-dependent protein–protein interactions. Oncogene 37, 5587–5604 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  121. Zhou, B. P. et al. HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat. Cell Biol. 3, 973–982 (2001).

    Article  PubMed  CAS  Google Scholar 

  122. Maurer, U., Charvet, C., Wagman, A. S., Dejardin, E. & Green, D. R. Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1. Mol. Cell 21, 749–760 (2006).

    Article  PubMed  CAS  Google Scholar 

  123. Datta, S. R. et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91, 231–241 (1997).

    Article  PubMed  CAS  Google Scholar 

  124. Fazakerley, D. J. et al. Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance. Nat. Commun. 14, 923 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  125. Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M. & Hemmings, B. A. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378, 785–789 (1995).

    Article  PubMed  CAS  Google Scholar 

  126. Cho, H. et al. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKBβ). Science 292, 1728–1731 (2001).

    Article  PubMed  CAS  Google Scholar 

  127. Jaiswal, N. et al. The role of skeletal muscle Akt in the regulation of muscle mass and glucose homeostasis. Mol. Metab. 28, 1–13 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  128. Kane, S. et al. A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain. J. Biol. Chem. 277, 22115–22118 (2002).

    Article  PubMed  CAS  Google Scholar 

  129. Ramm, G., Larance, M., Guilhaus, M. & James, D. E. A role for 14-3-3 in insulin-stimulated GLUT4 translocation through its interaction with the RabGAP AS160. J. Biol. Chem. 281, 29174–29180 (2006).

    Article  PubMed  CAS  Google Scholar 

  130. DiPilato, L. M. et al. The role of PDE3B phosphorylation in the inhibition of lipolysis by insulin. Mol. Cell. Biol. 35, 2752–2760 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  131. Gabbay, R. A. & Lardy, H. A. The antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action. FEBS Lett. 179, 7–11 (1985).

    Article  PubMed  CAS  Google Scholar 

  132. Luciano, A. K. et al. CLOCK phosphorylation by AKT regulates its nuclear accumulation and circadian gene expression in peripheral tissues. J. Biol. Chem. 293, 9126–9136 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  133. Hoxhaj, G. et al. Direct stimulation of NADP+ synthesis through Akt-mediated phosphorylation of NAD kinase. Science 363, 1088–1092 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  134. Love, N. R. et al. NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms. Proc. Natl Acad. Sci. USA 112, 1386–1391 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  135. Schmidlin, M. et al. The ARE-dependent mRNA-destabilizing activity of BRF1 is regulated by protein kinase B. EMBO J. 23, 4760–4769 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  136. Parker, R. & Sheth, U. P bodies and the control of mRNA translation and degradation. Mol. Cell 25, 635–646 (2007).

    Article  PubMed  CAS  Google Scholar 

  137. Larance, M. et al. Global phosphoproteomics identifies a major role for AKT and 14-3-3 in regulating EDC3. Mol. Cell. Proteomics 9, 682–694 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  138. Wei, W. et al. TRIM24 is an insulin-responsive regulator of P-bodies. Nat. Commun. 13, 3972 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  139. Sonnen, K. F. & Aulehla, A. Dynamic signal encoding—from cells to organisms. Semin. Cell Dev. Biol. 34, 91–98 (2014).

    Article  PubMed  Google Scholar 

  140. Kolch, W., Halasz, M., Granovskaya, M. & Kholodenko, B. N. The dynamic control of signal transduction networks in cancer cells. Nat. Rev. Cancer 15, 515–527 (2015).

    Article  PubMed  CAS  Google Scholar 

  141. Purvis, J. E. & Lahav, G. Encoding and decoding cellular information through signaling dynamics. Cell 152, 945–956 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  142. Kubota, H. & Kuroda, S. in Protein Modifications in Pathogenic Dysregulation of Signaling (eds Inoue, J. & Takekawa, M.) 95–109 (Springer, 2015).

  143. Vinayagam, A. et al. An integrative analysis of the InR/PI3K/Akt network identifies the dynamic response to insulin signaling. Cell Rep. 16, 3062–3074 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  144. Schmelzle, K., Kane, S., Gridley, S., Lienhard, G. E. & White, F. M. Temporal dynamics of tyrosine phosphorylation in insulin signaling. Diabetes 55, 2171–2179 (2006).

    Article  PubMed  CAS  Google Scholar 

  145. Kubota, H., Uda, S., Matsuzaki, F., Yamauchi, Y. & Kuroda, S. In vivo decoding mechanisms of the temporal patterns of blood insulin by the insulin–AKT pathway in the liver. Cell Syst. 7, 118–128 (2018).

    Article  PubMed  CAS  Google Scholar 

  146. Yang, G., Murashige, D. S., Humphrey, S. J. & James, D. E. A positive feedback loop between Akt and mTORC2 via SIN1 phosphorylation. Cell Rep. 12, 937–943 (2015).

    Article  PubMed  CAS  Google Scholar 

  147. Dillon, L. M. et al. P-REX1 creates a positive feedback loop to activate growth factor receptor, PI3K/AKT and MEK/ERK signaling in breast cancer. Oncogene 34, 3968–3976 (2015).

    Article  PubMed  CAS  Google Scholar 

  148. Copps, K. D. & White, M. F. Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2. Diabetologia 55, 2565–2582 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  149. Chin, J. E., Dickens, M., Tavare, J. M. & Roth, R. A. Overexpression of protein kinase C isoenzymes α, βI, γ, and ε in cells overexpressing the insulin receptor. Effects on receptor phosphorylation and signaling. J. Biol. Chem. 268, 6338–6347 (1993).

    Article  PubMed  CAS  Google Scholar 

  150. Knutson, V. P. Cellular trafficking and processing of the insulin receptor. FASEB J. 5, 2130–2138 (1991).

    Article  PubMed  CAS  Google Scholar 

  151. Lewis, R. E., Wu, G. P., MacDonald, R. G. & Czech, M. P. Insulin-sensitive phosphorylation of serine 1293/1294 on the human insulin receptor by a tightly associated serine kinase. J. Biol. Chem. 265, 947–954 (1990).

    Article  PubMed  CAS  Google Scholar 

  152. Pang, D. T., Sharma, B. R., Shafer, J. A., White, M. F. & Kahn, C. R. Predominance of tyrosine phosphorylation of insulin receptors during the initial response of intact cells to insulin. J. Biol. Chem. 260, 7131–7136 (1985).

    Article  PubMed  CAS  Google Scholar 

  153. Petersen, M. C. et al. Insulin receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance. J. Clin. Invest. 126, 4361–4371 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  154. Johnson, J. L. et al. An atlas of substrate specificities for the human serine/threonine kinome. Nature 613, 759–766 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  155. Goh, L. K. & Sorkin, A. Endocytosis of receptor tyrosine kinases. Cold Spring Harb. Perspect. Biol. 5, a017459 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  156. Hall, C., Yu, H. & Choi, E. Insulin receptor endocytosis in the pathophysiology of insulin resistance. Exp. Mol. Med. 52, 911–920 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  157. Song, R. et al. Central role of E3 ubiquitin ligase MG53 in insulin resistance and metabolic disorders. Nature 494, 375–379 (2013).

    Article  PubMed  CAS  Google Scholar 

  158. Holt, L. J. & Siddle, K. Grb10 and Grb14: enigmatic regulators of insulin action—and more? Biochem. J. 388, 393–406 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  159. Cooney, G. J. et al. Improved glucose homeostasis and enhanced insulin signalling in Grb14-deficient mice. EMBO J. 23, 582–593 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  160. Smith, F. M. et al. Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life. Mol. Cell. Biol. 27, 5871–5886 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  161. Kazi, J. U., Kabir, N. N., Flores-Morales, A. & Rönnstrand, L. SOCS proteins in regulation of receptor tyrosine kinase signaling. Cell. Mol. Life Sci. 71, 3297–3310 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  162. Yoneyama, Y. et al. Serine phosphorylation by mTORC1 promotes IRS-1 degradation through SCFβ-TRCP E3 ubiquitin ligase. iScience 5, 1–18 (2018).

    PubMed  PubMed Central  CAS  Google Scholar 

  163. Hançer, N. J. et al. Insulin and metabolic stress stimulate multisite serine/threonine phosphorylation of insulin receptor substrate 1 and inhibit tyrosine phosphorylation. J. Biol. Chem. 289, 12467–12484 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  164. Erion, D. M. & Shulman, G. I. Diacylglycerol-mediated insulin resistance. Nat. Med. 16, 400–402 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  165. Gehart, H., Kumpf, S., Ittner, A. & Ricci, R. MAPK signalling in cellular metabolism: stress or wellness? EMBO Rep. 11, 834–840 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  166. Zick, Y. Ser/Thr phosphorylation of IRS proteins: a molecular basis for insulin resistance. Sci. STKE 2005, pe4 (2005).

    Article  PubMed  Google Scholar 

  167. Batista, T. M., Haider, N. & Kahn, C. R. Defining the underlying defect in insulin action in type 2 diabetes. Diabetologia 64, 994–1006 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  168. Needham, E. J. et al. Personalized phosphoproteomics of skeletal muscle insulin resistance and exercise links MINDY1 to insulin action. Cell Metab. https://doi.org/10.1016/j.cmet.2024.10.020 (2024).

    Article  PubMed  Google Scholar 

  169. Lee, D. H. et al. Targeted disruption of ROCK1 causes insulin resistance in vivo. J. Biol. Chem. 284, 11776–11780 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  170. Furukawa, N. et al. Role of Rho-kinase in regulation of insulin action and glucose homeostasis. Cell Metab. 2, 119–129 (2005).

    Article  PubMed  CAS  Google Scholar 

  171. Copps, K. D. et al. Irs1 serine 307 promotes insulin sensitivity in mice. Cell Metab. 11, 84–92 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  172. Woo, J. R. et al. The serine phosphorylations in the IRS-1 PIR domain abrogate IRS-1 and IR interaction. Proc. Natl Acad. Sci. USA 121, e2401716121 (2024).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  173. Weigert, C. et al. The phosphorylation of Ser318 of insulin receptor substrate 1 is not per se inhibitory in skeletal muscle cells but is necessary to trigger the attenuation of the insulin-stimulated signal. J. Biol. Chem. 280, 37393–37399 (2005).

    Article  PubMed  CAS  Google Scholar 

  174. Waraich, R. S. et al. Phosphorylation of Ser357 of rat insulin receptor substrate-1 mediates adverse effects of protein kinase C-δ on insulin action in skeletal muscle cells. J. Biol. Chem. 283, 11226–11233 (2008).

    Article  PubMed  CAS  Google Scholar 

  175. Park, K. et al. Serine phosphorylation sites on IRS2 activated by angiotensin II and protein kinase C to induce selective insulin resistance in endothelial cells. Mol. Cell. Biol. 33, 3227–3241 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  176. Luo, M. et al. Identification of insulin receptor substrate 1 serine/threonine phosphorylation sites using mass spectrometry analysis: regulatory role of serine 1223. Endocrinology 146, 4410–4416 (2005).

    Article  PubMed  CAS  Google Scholar 

  177. Fritsche, L. et al. Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and mTOR: studies on the function of Ser675 and Ser907. Am. J. Physiol. Endocrinol. Metab. 300, E824–E836 (2011).

    Article  PubMed  CAS  Google Scholar 

  178. Tzatsos, A. Raptor binds the SAIN (Shc and IRS-1 NPXY binding) domain of insulin receptor substrate-1 (IRS-1) and regulates the phosphorylation of IRS-1 at Ser-636/639 by mTOR. J. Biol. Chem. 284, 22525–22534 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  179. Tiganis, T. PTP1B and TCPTP—nonredundant phosphatases in insulin signaling and glucose homeostasis. FEBS J. 280, 445–458 (2013).

    Article  PubMed  CAS  Google Scholar 

  180. Wu, X. et al. Mammalian sprouty proteins assemble into large monodisperse particles having the properties of intracellular nanobatteries. Proc. Natl Acad. Sci. USA 102, 14058–14062 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  181. Montagner, A. et al. A novel role for Gab1 and SHP2 in epidermal growth factor-induced Ras activation. J. Biol. Chem. 280, 5350–5360 (2005).

    Article  PubMed  CAS  Google Scholar 

  182. Yadav, Y. & Dey, C. S. Ser/Thr phosphatases: one of the key regulators of insulin signaling. Rev. Endocr. Metab. Disord. 23, 905–917 (2022).

    Article  PubMed  CAS  Google Scholar 

  183. He, R.-J., Yu, Z.-H., Zhang, R.-Y. & Zhang, Z.-Y. Protein tyrosine phosphatases as potential therapeutic targets. Acta Pharmacol. Sin. 35, 1227–1246 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  184. Shi, Y. Serine/threonine phosphatases: mechanism through structure. Cell 139, 468–484 (2009).

    Article  PubMed  CAS  Google Scholar 

  185. Lake, D., Corrêa, S. A. L. & Müller, J. Negative feedback regulation of the ERK1/2 MAPK pathway. Cell. Mol. Life Sci. 73, 4397–4413 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  186. Dougherty, M. K. et al. Regulation of Raf-1 by direct feedback phosphorylation. Mol. Cell 17, 215–224 (2005).

    Article  PubMed  CAS  Google Scholar 

  187. Rozakis-Adcock, M., van der Geer, P., Mbamalu, G. & Pawson, T. MAP kinase phosphorylation of mSos1 promotes dissociation of mSos1–Shc and mSos1–EGF receptor complexes. Oncogene 11, 1417–1426 (1995).

    PubMed  CAS  Google Scholar 

  188. Caunt, C. J., Armstrong, S. P., Rivers, C. A., Norman, M. R. & McArdle, C. A. Spatiotemporal regulation of ERK2 by dual specificity phosphatases. J. Biol. Chem. 283, 26612–26623 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  189. Zimmermann, S. & Moelling, K. Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 286, 1741–1744 (1999).

    Article  PubMed  CAS  Google Scholar 

  190. Lynch, D. K. & Daly, R. J. PKB-mediated negative feedback tightly regulates mitogenic signalling via Gab2. EMBO J. 21, 72–82 (2002).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  191. Miyazaki, M. & Takemasa, T. TSC2/Rheb signaling mediates ERK-dependent regulation of mTORC1 activity in C2C12 myoblasts. FEBS Open Bio 7, 424–433 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  192. Carriere, A. et al. ERK1/2 phosphorylate Raptor to promote Ras-dependent activation of mTOR complex 1 (mTORC1). J. Biol. Chem. 286, 567–577 (2011).

    Article  PubMed  CAS  Google Scholar 

  193. Ebi, H. et al. PI3K regulates MEK/ERK signaling in breast cancer via the Rac-GEF, P-Rex1. Proc. Natl Acad. Sci. USA 110, 21124–21129 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  194. Higuchi, M., Onishi, K., Kikuchi, C. & Gotoh, Y. Scaffolding function of PAK in the PDK1–Akt pathway. Nat. Cell Biol. 10, 1356–1364 (2008).

    Article  PubMed  CAS  Google Scholar 

  195. Hawley, S. A. et al. Phosphorylation by Akt within the ST loop of AMPK-α1 down-regulates its activation in tumour cells. Biochem. J. 459, 275–287 (2014).

    Article  PubMed  CAS  Google Scholar 

  196. Inoki, K., Zhu, T. & Guan, K.-L. TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590 (2003).

    Article  PubMed  CAS  Google Scholar 

  197. Gwinn, D. M. et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell 30, 214–226 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  198. Needham, E. J. et al. Personalized phosphoproteomics identifies functional signaling. Nat. Biotechnol. 40, 576–584 (2022).

    Article  PubMed  CAS  Google Scholar 

  199. Kazyken, D. et al. AMPK directly activates mTORC2 to promote cell survival during acute energetic stress. Sci. Signal. 12, eaav3249 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  200. Accili, D. & Arden, K. C. FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell 117, 421–426 (2004).

    Article  PubMed  CAS  Google Scholar 

  201. Baker, S. A. & Rutter, J. Metabolites as signalling molecules. Nat. Rev. Mol. Cell Biol. 24, 355–374 (2023).

    Article  PubMed  CAS  Google Scholar 

  202. Trefely, S. et al. Kinome screen identifies PFKFB3 and glucose metabolism as important regulators of the insulin/insulin-like growth factor (IGF)-1 signaling pathway. J. Biol. Chem. 290, 25834–25846 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  203. Cong, Y. et al. Tripartite-motif 3 represses ovarian cancer progression by downregulating lactate dehydrogenase A and inhibiting AKT signaling. Mol. Cell. Biochem. 479, 3405–3424 (2024).

    Article  PubMed  CAS  Google Scholar 

  204. Xu, K. et al. Glycolysis fuels phosphoinositide 3-kinase signaling to bolster T cell immunity. Science 371, 405–410 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  205. Jani, S. et al. Distinct mechanisms involving diacylglycerol, ceramides, and inflammation underlie insulin resistance in oxidative and glycolytic muscles from high fat-fed rats. Sci. Rep. 11, 19160 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  206. Fox, T. E. et al. Ceramide recruits and activates protein kinase C ζ (PKCζ) within structured membrane microdomains. J. Biol. Chem. 282, 12450–12457 (2007).

    Article  PubMed  CAS  Google Scholar 

  207. Ghosh, N. et al. Ceramide-activated protein phosphatase involvement in insulin resistance via Akt, serine/arginine-rich protein 40, and ribonucleic acid splicing in L6 skeletal muscle cells. Endocrinology 148, 1359–1366 (2007).

    Article  PubMed  CAS  Google Scholar 

  208. Diaz-Vegas, A. et al. Mitochondrial electron transport chain, ceramide, and coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle. eLife 12, RP87340 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  209. Judge, S. et al. Analysis of multiple insulin actions in single muscle fibers from insulin-resistant mice reveals selective defect in endogenous GLUT4 translocation. Diabetes https://doi.org/10.2337/db25-0022 (2025).

    Article  PubMed  Google Scholar 

  210. Myers, D. R., Zikherman, J. & Roose, J. P. Tonic signals: why do lymphocytes bother? Trends Immunol. 38, 844–857 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  211. Eggert, J. & Au-Yeung, B. B. Functional heterogeneity and adaptation of naive T cells in response to tonic TCR signals. Curr. Opin. Immunol. 73, 43–49 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  212. Kjærgaard, J. et al. Personalized molecular signatures of insulin resistance and type 2 diabetes. Cell 188, 4106–4122.e16 (2025).

    Article  PubMed  Google Scholar 

  213. Batista, T. M. et al. A cell-autonomous signature of dysregulated protein phosphorylation underlies muscle insulin resistance in type 2 diabetes. Cell Metab. 32, 844–859 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  214. Haider, N. et al. Signaling defects associated with insulin resistance in nondiabetic and diabetic individuals and modification by sex. J. Clin. Invest. 131, e151818 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  215. Tonks, K. T. et al. Impaired Akt phosphorylation in insulin-resistant human muscle is accompanied by selective and heterogeneous downstream defects. Diabetologia 56, 875–885 (2013).

    Article  PubMed  CAS  Google Scholar 

  216. Yu, J. et al. JNK3 signaling pathway activates ceramide synthase leading to mitochondrial dysfunction. J. Biol. Chem. 282, 25940–25949 (2007).

    Article  PubMed  CAS  Google Scholar 

  217. Sánchez-Aguilera, P. et al. Role of ABCA1 on membrane cholesterol content, insulin-dependent Akt phosphorylation and glucose uptake in adult skeletal muscle fibers from mice. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1863, 1469–1477 (2018).

    Article  PubMed  Google Scholar 

  218. Tan, S.-X. et al. Selective insulin resistance in adipocytes. J. Biol. Chem. 290, 11337–11348 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  219. Tan, S.-X. et al. Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes. J. Biol. Chem. 287, 6128–6138 (2012).

    Article  PubMed  CAS  Google Scholar 

  220. Diaz-Vegas, A. et al. A high-content endogenous GLUT4 trafficking assay reveals new aspects of adipocyte biology. Life Sci. Alliance 6, e202201585 (2023).

    Article  PubMed  CAS  Google Scholar 

  221. van Gerwen, J. et al. The genetic and dietary landscape of the muscle insulin signalling network. eLife 12, RP89212 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  222. Deprez, J., Vertommen, D., Alessi, D. R., Hue, L. & Rider, M. H. Phosphorylation and activation of heart 6-phosphofructo-2-kinase by protein kinase B and other protein kinases of the insulin signaling cascades. J. Biol. Chem. 272, 17269–17275 (1997).

    Article  PubMed  CAS  Google Scholar 

  223. Marsin, A. S. et al. Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia. Curr. Biol. 10, 1247–1255 (2000).

    Article  PubMed  CAS  Google Scholar 

  224. Stenbit, A. E. et al. GLUT4 heterozygous knockout mice develop muscle insulin resistance and diabetes. Nat. Med. 3, 1096–1101 (1997).

    Article  PubMed  CAS  Google Scholar 

  225. Zisman, A. et al. Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance. Nat. Med. 6, 924–928 (2000).

    Article  PubMed  CAS  Google Scholar 

  226. Diaz-Vegas, A. et al. Deletion of miPEP in adipocytes protects against obesity and insulin resistance by boosting muscle metabolism. Mol. Metab. https://doi.org/10.1016/j.molmet.2024.101983 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  227. Brown, M. S. & Goldstein, J. L. Selective versus total insulin resistance: a pathogenic paradox. Cell Metab. 7, 95–96 (2008).

    Article  PubMed  CAS  Google Scholar 

  228. Lotta, L. A. et al. Integrative genomic analysis implicates limited peripheral adipose storage capacity in the pathogenesis of human insulin resistance. Nat. Genet. 49, 17–26 (2017).

    Article  PubMed  CAS  Google Scholar 

  229. Semple, R. K., Savage, D. B., Cochran, E. K., Gorden, P. & O’Rahilly, S. Genetic syndromes of severe insulin resistance. Endocr. Rev. 32, 498–514 (2011).

    Article  PubMed  CAS  Google Scholar 

  230. Møller, G. et al. The role of a traditional and western diet on glucose homeostasis in Greenlandic Inuit carriers and non-carriers of type 2 diabetes variant in the TBC1D4 gene: a protocol for a randomized clinical trial. Contemp. Clin. Trials Commun. 21, 100734 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  231. Barbieri, M., Bonafè, M., Franceschi, C. & Paolisso, G. Insulin/IGF-I-signaling pathway: an evolutionarily conserved mechanism of longevity from yeast to humans. Am. J. Physiol. Endocrinol. Metab. 285, E1064–E1071 (2003).

    Article  PubMed  CAS  Google Scholar 

  232. Kenyon, C. The first long-lived mutants: discovery of the insulin/IGF-1 pathway for ageing. Philos. Trans. R. Soc. Lond. B Biol. Sci. 366, 9–16 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  233. Boura-Halfon, S. & Zick, Y. Phosphorylation of IRS proteins, insulin action, and insulin resistance. Am. J. Physiol. Endocrinol. Metab. 296, E581–E591 (2009).

    Article  PubMed  CAS  Google Scholar 

  234. Yu, Y. et al. Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling. Science 332, 1322–1326 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  235. Loh, K. et al. Insulin controls food intake and energy balance via NPY neurons. Mol. Metab. 6, 574–584 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  236. Schwartz, M. W. et al. Cooperation between brain and islet in glucose homeostasis and diabetes. Nature 503, 59–66 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  237. Ono, H. et al. Activation of hypothalamic S6 kinase mediates diet-induced hepatic insulin resistance in rats. J. Clin. Invest. 118, 2959–2968 (2008).

    PubMed  PubMed Central  CAS  Google Scholar 

  238. De Felice, F. G. Alzheimer’s disease and insulin resistance: translating basic science into clinical applications. J. Clin. Invest. 123, 531–539 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  239. Makhijani, P. et al. Regulation of the immune system by the insulin receptor in health and disease. Front. Endocrinol. 14, 1128622 (2023).

    Article  Google Scholar 

  240. Case, L. B., Zhang, X., Ditlev, J. A. & Rosen, M. K. Stoichiometry controls activity of phase-separated clusters of actin signaling proteins. Science 363, 1093–1097 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  241. Lin, C.-W. et al. A two-component protein condensate of the EGFR cytoplasmic tail and Grb2 regulates Ras activation by SOS at the membrane. Proc. Natl Acad. Sci. USA 119, e2122531119 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  242. Dall’Agnese, A. et al. The dynamic clustering of insulin receptor underlies its signaling and is disrupted in insulin resistance. Nat. Commun. 13, 7522 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  243. Kim, S. J. et al. mTOR complex 2 regulates proper turnover of insulin receptor substrate-1 via the ubiquitin ligase subunit Fbw8. Mol. Cell 48, 875–887 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  244. Kono, T. & Barham, F. W. The relationship between the insulin-binding capacity of fat cells and the cellular response to insulin. Studies with intact and trypsin-treated fat cells. J. Biol. Chem. 246, 6210–6216 (1971).

    Article  PubMed  CAS  Google Scholar 

  245. Cleasby, M. E., Reinten, T. A., Cooney, G. J., James, D. E. & Kraegen, E. W. Functional studies of Akt isoform specificity in skeletal muscle in vivo; maintained insulin sensitivity despite reduced insulin receptor substrate-1 expression. Mol. Endocrinol. 21, 215–228 (2007).

    Article  PubMed  CAS  Google Scholar 

  246. Le Marchand-Brustel, Y., Jeanrenaud, B. & Freychet, P. Insulin binding and effects in isolated soleus muscle of lean and obese mice. Am. J. Physiol. 234, E348–E358 (1978).

    PubMed  Google Scholar 

  247. Rice, K. M., Lienhard, G. E. & Garner, C. W. Regulation of the expression of pp160, a putative insulin receptor signal protein, by insulin, dexamethasone, and 1-methyl-3-isobutylxanthine in 3T3-L1 adipocytes. J. Biol. Chem. 267, 10163–10167 (1992).

    Article  PubMed  CAS  Google Scholar 

  248. Hoehn, K. L. et al. IRS1-independent defects define major nodes of insulin resistance. Cell Metab. 7, 421–433 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

D.E.J. is an Australian Research Council Laureate Fellow. J.G.B., D.E.J. and A.D.-V. are supported by a National Health and Medical Research Council grant (GNT2013621). We are extremely grateful to J. Stoeckli for the thoughtful discussion of our work and to B. Parker, L. Nguyen, S. Masson and H. Cutler for carefully reading and commenting on the manuscript.

Author information

Author notes

  1. These authors contributed equally: James G. Burchfield, Alexis Diaz-Vegas.

Authors and Affiliations

  1. School of Life and Environmental Sciences, University of Sydney, Camperdown, New South Wales, Australia

    James G. Burchfield, Alexis Diaz-Vegas & David E. James

  2. Charles Perkins Centre, University of Sydney, Camperdown, New South Wales, Australia

    James G. Burchfield, Alexis Diaz-Vegas & David E. James

  3. School of Medical Sciences, University of Sydney, Camperdown, New South Wales, Australia

    David E. James

Authors
  1. James G. Burchfield
    View author publications

    Search author on:PubMed Google Scholar

  2. Alexis Diaz-Vegas
    View author publications

    Search author on:PubMed Google Scholar

  3. David E. James
    View author publications

    Search author on:PubMed Google Scholar

Contributions

All authors contributed equally to all aspects of the article.

Corresponding author

Correspondence to David E. James.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Metabolism thanks Shaodong Guo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Yanina-Yasmin Pesch, in collaboration with the Nature Metabolism team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Burchfield, J.G., Diaz-Vegas, A. & James, D.E. The insulin signalling network. Nat Metab 7, 1745–1764 (2025). https://doi.org/10.1038/s42255-025-01349-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/s42255-025-01349-z

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing