STATE-OF-THE-ART REVIEW IN ENDOCRINOLOGY
The role of insulin signaling with FOXO and FOXK transcription factors
2024 Volume 71 Issue 10 Pages 939-944
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2024 Volume 71 Issue 10 Pages 939-944
Insulin is an essential hormone for animal activity and survival, and it controls the metabolic functions of the entire body. Throughout the evolution of metazoan animals and the development of their brains, a sustainable energy supply has been essential to overcoming the competition for survival under various environmental stresses. Managing energy for metabolism, preservation, and consumption inevitably involves high oxidative stress, causing tissue damage in various organs. In both mice and humans, excessive dietary intake can lead to insulin resistance in various organs, ultimately displaying metabolic syndrome and type 2 diabetes. Insulin signals require thorough regulation to maintain metabolism across diverse environments. Recent studies demonstrated that two types of forkhead-box family transcription factors, FOXOs and FOXKs, are related to the switching of insulin signals during fasting and feeding states. Insulin signaling plays a role in supporting higher activity during periods of sufficient food supply and in promoting survival during times of insufficient food supply. The insulin receptor depends on the tyrosine phosphatase feedback of insulin signaling to maintain adipocyte insulin responsiveness. α4, a regulatory subunit of protein phosphatase 2A (PP2A), has been shown to play a crucial role in modulating insulin signaling pathways by regulating the phosphorylation status of key proteins involved in these pathways. This short review summarizes the current understanding of the molecular mechanism related to the regulation of insulin signals.
Insulin interaction with insulin receptors (InsR) triggers subsequent structural changes and autophosphorylation of three consecutive tyrosine residues, Tyr1158, Tyr1162, and Tyr1163, within the intracellular tyrosine kinase domain, augmenting kinase activity, and inducing the subsequent phosphorylation of IRS proteins (such as IRS1, IRS2, IRS3, and IRS4 [1]). These IRS proteins possess pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains crucial for membrane and receptor binding, along with multiple phosphorylation sites that regulate their stability and function. Upon insulin stimulation, IRS proteins bind to a phosphorylated NPEpY972 motif located in the juxtamembrane region of the InsR [2], facilitating the phosphorylation of tyrosine residues within IRS tails, enabling the recruitment of downstream signaling proteins, including the p85 regulatory subunit of Class 1A phosphoinositide 3-kinase (PI3K), crucial for metabolic signaling [3]. Additionally, this binding results in the formation of phosphatidylinositol 3,4,5-bisphosphate (PIP3) that recruits PDK1 and activates serine/threonine kinase AKT. Consequently, AKT phosphorylates various substrate proteins—leading to Glut 4-mediated glucose uptake, GSK3-mediated glycogen synthesis, and mTOR-mediated protein synthesis—and regulating various gene transcriptions through transcription factor forkhead protein, FOXOs. The rapamycin complex (mTORC) exists in two distinct forms, mTORC1 and mTORC2 [4]. While mTORC1 promotes cell growth and proliferation by regulating processes such as protein synthesis [5], mTORC2 is implicated in mRNA processing via IMP1 phosphorylation and AKT activation [6]. Another major pathway through IRS-1 and Shc activates Grb2-Sos-Ras-Raf-MEK evoking MAPK activation, which regulates cell proliferation, growth, and differentiation (Fig. 1).
Autophosphorylation of the receptors lead to the phosphorylation of receptor substrates such as IRS and Shc proteins. While Shc activates the Ras-Raf-MAPK pathway, IRS proteins predominantly activate the PI3K-Akt pathway by recruiting and activating PI3K, leading to the generation of the second messenger PIP3. Membrane-bound PIP3 recruits and activates PDK-1, which then activates Akt, regulating glucose uptake, protein synthesis and glycogen synthesis.
Fig. 1 is generated with BioRender (https://www.biorender.com).
These signaling cascades have developed through animal evolution, and here a study using a lower animal model has revealed an intriguing aspect of insulin signaling. An insulin receptor-like gene called daf-2 (the worm insulin receptor homolog) in Caenorhabditis (C.) elegans mediates endocrine signaling and is involved in metabolic and developmental changes. Interestingly, decreased daf-2 signaling in worms has been associated with enhanced stress resistance and extended lifespan. However, when the daf-2 mutant worms are mixed with wild-type worms under unstressed conditions, the daf-2 mutant worms are outcompeted by the wild-type worms. This study suggests a significant fitness cost associated with reduced daf-2 activity, impacting both fertility and longevity [7]. These pathways presumably orchestrate various cellular processes essential for mediating metabolic regulation, growth effects, and longevity by minimizing stress in the body.
Insulin and insulin-like growth factor (IGF-1) mediate their biological effects through InsR and IGF-1 receptors (IGF-1R), respectively. These receptors belong to a family of highly homologous tyrosine kinase receptors. InsR and IGF-1R are composed of tetrameric proteins, consisting of two extracellular α subunits and two membrane-spanning β subunits connected by disulfide bonds. InsR messenger RNA (mRNA) generates two distinct isoforms, Isoform A and Isoform B, with differential expression patterns particularly in fetal tissues and the brain [8]. While insulin and IGF-1 primarily bind to their respective receptors, they can also interact with alternative receptors with lower affinity [9]. This interaction induces conformational changes in InsR and IGF-1R, triggering the kinase activity of the β subunits [10-12]. By binding to their respective receptors on the cell surface, InsR and IGF-1R regulate key control functions of metabolism, growth, and mitogenesis [13]. The activation of these receptors results in diverse physiological outcomes, with InsR primarily regulating metabolic functions and IGF-1R participating in growth and mitogenesis. InsR tends to phosphorylate IRS proteins, whereas IGF-1R prefers the phosphorylation of Shc, a protein containing src homology 2 (SH2) domains [14]. Notably, a significant sequence disparity close to the NPEY motif in the juxtamembrane region distinguishes InsR and IGF-1R. This difference, particularly at residue 973 (leucine in InsR and phenylalanine in IGF-1R), affects the receptors’ preferences in binding to downstream signaling molecules [14, 15].
A crucial target of AKT is the forkhead box O (FOXO) family of transcription factors. FOX proteins comprise a superfamily of highly conserved transcription factors characterized by the presence of the evolutionarily conserved “forkhead” or “winged-helix” DNA-binding domain (DBD), facilitating specific binding to the conserved sequence 5'-TTGTTTAC-3' [16]. Presently, more than 54 FOX family members have been identified in mammals, with over 50 in the human genome alone. This family is further subdivided into 19 subfamilies (from FOXA to FOXS) based on sequence similarity [17]. The FOXO transcription factor family, consisting of FOXO1, FOXO3, FOXO4, and FOXO6, shares homology with C. elegans DAF-16, known for its role in doubling lifespan upon loss of daf-2 [18]. FOXOs play a pivotal role in regulating adaptation to nutrient availability stress, mirroring their involvement in stress response and longevity regulation in C. elegans [19]. In mammals, FOXO family members regulate various cellular processes, particularly FOXO1, FOXO3, and FOXO4, while FOXO6 exhibits distinct features, notably its apparent inability to translocate between the nucleus and cytoplasm.
Insulin stimulation triggers the phosphorylation of FOXOs by AKT, leading to their exclusion from the nucleus and subsequent suppression of their target genes. This leads to decreased gluconeogenesis in the liver, reduced autophagy and protein degradation in muscles, increased adipocyte differentiation, and reduced IGFBP1 expression in the liver—consequently enhancing the biological availability of circulating IGF-1 and promoting overall body growth [20-22]. Due to the negative regulation of FOXO-regulated gene expression, FOXO deletion can reverse many metabolic abnormalities induced by insulin resistance stemming from InsR/IGF-1R deletion or Akt1/Akt2 deletion [23, 24].
FOXOs primarily regulate glucose metabolism through the expression of gluconeogenic enzymes, ensuring glucose availability during fasting or exercise. In the liver, FOXOs promote gluconeogenic enzyme expression, such as pyruvate dehydrogenase kinase 4 (PDK4) and glucose-6-phosphatase, facilitating glucose release into the bloodstream. Active FOXOs in the liver induce ApoC3 transcription, leading to elevated plasma triglyceride levels [25]. The action of FOXO transcription factors can result in the activation or repression of gene expression. While insulin signaling downregulates various FOXO-dependent genes, it can also upregulate others, such as glucokinase (Gck) [26]. FoxO1 associates with the corepressor SIN3 transcription regulator family member A (SIN3A) to downregulate Gck expression and decrease lipogenesis [27]. In muscles, FOXOs facilitate the metabolic switch from glucose metabolism to lipid oxidation. Notably, FOXOs also regulate mitochondrial protein expression, influencing mitochondrial dynamics and metabolism [28]. They inhibit mitochondrial division by suppressing mitochondrial fission, contributing to mitochondrial homeostasis [28].
Another emerging subset within the FOX protein family involved in insulin signaling modulation comprises the transcription factors FOXK1 and FOXK2. In contrast to FOXOs, FOXKs are activated by insulin, leading to increased nuclear localization and transcriptional activity. Regulation of FOXKs is intricate: in the basal state GSK3 phosphorylates FOXKs, increasing interaction with 14-3-3 proteins and leading to cytoplasmic exclusion [29, 30]. This phosphorylation is counteracted upon activation of Akt and mTORC1 by insulin. FOXK1 and FOXK2 share two crucial domains, FOX (forkhead winged helix-turn-helix DNA-binding domain) and FHA (forkhead-associated domain), which facilitate protein interactions and govern cell cycle dynamics. These domains exhibit significant conservation between FOXK1 and FOXK2.
The FOX domain of FOXK1 facilitates direct DNA interaction, while the FHA domain mediates interactions with suppressor of defective silencing 3 (Sds3) [31]. Moreover, the N-terminal region harbors the Swi-independent 3b-interacting domain (SID), promoting direct interaction between FOXK1 and Sin3 [32]. For FOXK2, the 54–171 amino acid region containing the FHA domain is crucial for interaction with dishevelled (DVL) and Sds3 [33]. The FHA domain is required for interaction with BRCA1-associated ring domain 1 (BARD1) [34]. FOXK1/2 prominently recruit the Sin3A-HDAC complex, specifically modulating the expression of downstream target genes crucial for atrophy and autophagy, including Tcfap4, Junb, Ccne2, and Fbxo32 [35]. Nutrient restriction prompts the translocation of FOXK1 to the cytoplasm, accompanied by increased expression of these genes. FOXK1 plays a pivotal role in skeletal muscle regeneration [36] and controls the cell-cycle progression of myogenic progenitors by repressing the expression of p21 [37]. Additionally, both FOXK1 and FOXK2 stimulate aerobic glycolysis by upregulating the expression of key glycolytic enzymes such as hexokinase-2, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in muscle [38]. Moreover, FOXK1 is recruited to insulin response sequences in the promoters of phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase (G6pc) genes in hepatocytes upon insulin stimulation, acting as a transcriptional repressor [39]. FoxK1 and FoxO proteins may exhibit complementary regulatory effects (Fig. 2). Consequently, insulin action leads to the exclusion of FoxO1 from the nucleus, while the opposite is observed for FoxK1. Furthermore, FoxO1 and FoxK1 binding sites overlap with numerous genes. However, Allu et al. recently showed that FoxK1 and FoxO1 also interact with numerous other genes by ChIP-seq analysis, suggesting that their regulatory effects are complementary [40]. These findings contribute to a comprehensive understanding of the downstream effects of insulin signaling mediated by FOXK transcription factors, highlighting their diverse roles in metabolic regulation.
Following insulin stimulation, FOXOs are phosphorylated by Akt, leading to their interaction with 14-3-3 proteins and translocation from the nucleus to the cytoplasm. Conversely, FOXKs are inhibited by GSK3-mediated phosphorylation, resulting in their translocation from the membrane and cytoplasm to the nucleus.
Fig. 2 is generated with BioRender (https://www.biorender.com).
Central to InsR signaling is the dynamic regulation of its phosphorylation status, which is finely controlled by the opposing actions of kinases and phosphatases. While phosphorylation of InsR initiates downstream signaling cascades, its dephosphorylation is crucial for terminating insulin signaling and maintaining cellular homeostasis. Dephosphorylation of InsR primarily occurs through the activity of protein phosphatases, particularly protein tyrosine phosphatase 1B (PTP1B), which catalyze the removal of phosphate groups from tyrosine residues within the InsR kinase domain and substrate docking sites. PTP1B plays a pivotal role in insulin action as evidenced by studies using PTP1B knockout (KO) mice, which exhibit enhanced insulin sensitivity, elevated insulin receptor phosphorylation, and resistance to obesity and insulin resistance [41, 42]. Conversely, the activities of downstream protein kinases like Akt, PKC, S6K, and extracellular signal-regulated kinase (ERK) are under the regulation of serine/threonine protein phosphatase 2A (PP2A), constituting approximately 80% of serine/threonine phosphatase activity in cells [43]. Some studies suggest an overactivation of PP2A in diabetic conditions. Recent findings highlight the interplay between PP2A and PTP1B facilitated by α4, a protector of the catalytic subunit of PP2A in adipose tissues [44]. This mechanism establishes a feedback loop governing insulin receptor signaling, modulating tyrosine phosphorylation status from downstream to upstream. Adipocyte-specific deletion of α4 in mice disrupts insulin-induced Akt-mediated serine/threonine phosphorylation and diminishes insulin-induced InsR tyrosine phosphorylation by attenuating the interaction between α4 and Y-box protein 1 (YBX1), consequently enhancing the expression of the tyrosine phosphatase PTP1B [44] (Fig. 3).
Dephosphorylation of InsR is facilitated by the action of protein tyrosine phosphatase 1B (PTP1B). α4, serving as a regulatory subunit of PP2A, modulates the activity of the transcription factor Y-box protein 1 (YBX1) associated with PTP1B. This mechanism forms a feedback loop that controls the insulin receptor signaling pathway, influencing the status of downstream-to-upstream tyrosine phosphorylation.
Fig. 3 is generated with BioRender (https://www.biorender.com).
Insulin and IGF signaling pathways play pivotal roles in metabolism, growth, and development across species. Through decades of research, we have gained profound insights into the molecular mechanisms governing these pathways, highlighting their evolutionary conservation and critical roles in cellular homeostasis. Key regulators such as FOXO and FOXK transcription factors modulate insulin responses, impacting glucose homeostasis and metabolic functions. Additionally, protein phosphatases like PTP1B and PP2A tightly regulate insulin signaling dynamics. Understanding these pathways and the mechanisms underlying insulin resistance is essential for developing therapies for type 2 diseases. Furthermore, translating our understanding of these pathways into new therapies for insulin resistance-related diseases is a critical challenge for the next decade, promising to advance our ability to manage diabetes.
The author was supported by JSPS KAKENHI (JP 21K08532).
The author declares that he has no conflicts of interest associated with this research.
