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Phosphorylation of IRS-1 at Ser-1101 causes inhibition of insulin signaling. Serum-deprived CHO cells expressing either HA-IRS-1 or HA-IRS-1 (S1101A) were incubated either in an amino acid-deprived or amino acidcontaining medium for 1 h and stimulated or not with 100 nM insulin for the last 30 min of incubation as indicated. (A and B) Phosphorylation of IRS-1 was determined in anti-HA immunoprecipitates by using phosphospecific antibodies against IRS-1 Ser-307, Ser-312, Ser-636/639, and Ser-1101 (A) and phosphotyrosine (B). Levels of HA-IRS-1 recovered in anti-HA immunoprecipitates also are shown. (C) Phosphorylation and expression of Akt were determined in whole-cell extracts by using antibodies against phospho-Akt Ser-473 (Upper) and Akt (Lower). Results are of one representative experiment repeated at least three times. (D-F) Quantification of IRS-1 Ser-1101 (D), IRS-1 Tyr (E), and Akt Ser-473 (F) phosphorylation. * , P 0.05 vs. corresponding CHO cells expressing wild-type HA-IRS-1.
Source publication
S6K1 has emerged as a critical signaling component in the development of insulin resistance through phosphorylation and inhibition of IRS-1 function. This effect can be triggered directly by nutrients such as amino acids or by insulin through a homeostatic negative-feedback loop. However, the role of S6K1 in mediating IRS-1 phosphorylation in a phy...
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... ability of insulin to induce tyrosine phosphorylation of either an HA epitope- tagged wild-type IRS-1 (HA-IRS-1) or an IRS-1 S1101A (HA- IRS-1 S1101A) mutant after their transfection in CHO cells. Incubation of CHO cells with amino acids and insulin increased the phosphorylation of HA-IRS-1 on Ser-1101, but not that of the HA-IRS-1 S1101A mutant (Fig. 4 A and D). Importantly, phos- phorylation of IRS-1 at Ser-307, Ser-312, and Ser-636/639 (equiv- alent to mouse Ser-302, Ser-307, and Ser-632/635) appeared to be unaffected by the S1101A mutation of HA-IRS-1 ( Fig. 4A and SI Fig. 8). We further observed that insulin-induced tyrosine phos- phorylation of HA-IRS-1 S1101A in the presence of amino ...
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... cells with amino acids and insulin increased the phosphorylation of HA-IRS-1 on Ser-1101, but not that of the HA-IRS-1 S1101A mutant (Fig. 4 A and D). Importantly, phos- phorylation of IRS-1 at Ser-307, Ser-312, and Ser-636/639 (equiv- alent to mouse Ser-302, Ser-307, and Ser-632/635) appeared to be unaffected by the S1101A mutation of HA-IRS-1 ( Fig. 4A and SI Fig. 8). We further observed that insulin-induced tyrosine phos- phorylation of HA-IRS-1 S1101A in the presence of amino acids was significantly elevated, compared with that of wild-type HA- IRS-1 (Fig. 4 B and E). These findings indicate that phosphorylation of Ser-1101 by S6K1 is inhibitory to insulin receptor signaling. Consistent with ...
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... and Ser-636/639 (equiv- alent to mouse Ser-302, Ser-307, and Ser-632/635) appeared to be unaffected by the S1101A mutation of HA-IRS-1 ( Fig. 4A and SI Fig. 8). We further observed that insulin-induced tyrosine phos- phorylation of HA-IRS-1 S1101A in the presence of amino acids was significantly elevated, compared with that of wild-type HA- IRS-1 (Fig. 4 B and E). These findings indicate that phosphorylation of Ser-1101 by S6K1 is inhibitory to insulin receptor signaling. Consistent with this conclusion, we found that Akt Ser-473 phos- phorylation, a downstream effector of PI3-kinase, was enhanced in cells expressing the S1101A-mutated form of IRS-1, compared with those expressing its ...
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... These findings indicate that phosphorylation of Ser-1101 by S6K1 is inhibitory to insulin receptor signaling. Consistent with this conclusion, we found that Akt Ser-473 phos- phorylation, a downstream effector of PI3-kinase, was enhanced in cells expressing the S1101A-mutated form of IRS-1, compared with those expressing its wild-type counterpart (Fig. 4 C and F). We previously showed that S6K1 Thr-389 phosphorylation in the liver of diet-induced obese rats is markedly elevated in the fasting state and that this response is accelerated in the presence of insulin, peaking 5 min after injection (26). Here we determined whether genetically induced obesity in mice is associated with S6K1 activa- ...
Citations
... In this context, it is relevant to mention that others have presented evidence for a heterodimeric interaction between insulin receptor and MET (Fafalios et al, 2011), which could be involved in the HGF independent basal phosphorylation of MET in WD mice. This heterodimer and the known interactions between MET and the insulin receptor substrate 1 and 2 (IRS1 and IRS2) (Fafalios et al, 2011) (DeAngelis et al, 2010 have not been explicitly included in our model, but could be integrated in the future to further elucidate effects resulting in the observed downregulation of AKT as IRS1 is a target protein of the negative feedback regulation via S6K (Tremblay et al, 2007;Zhang et al, 2008). ...
Chronic liver diseases are worldwide on the rise. Due to the rapidly increasing incidence, in particular in Western countries, metabolic dysfunction-associated steatotic liver disease (MASLD) is gaining importance as the disease can develop into hepatocellular carcinoma. Lipid accumulation in hepatocytes has been identified as the characteristic structural change in MASLD development, but molecular mechanisms responsible for disease progression remained unresolved. Here, we uncover in primary hepatocytes from a preclinical model fed with a Western diet (WD) an increased basal MET phosphorylation and a strong downregulation of the PI3K-AKT pathway. Dynamic pathway modeling of hepatocyte growth factor (HGF) signal transduction combined with global proteomics identifies that an elevated basal MET phosphorylation rate is the main driver of altered signaling leading to increased proliferation of WD-hepatocytes. Model-adaptation to patient-derived hepatocytes reveal patient-specific variability in basal MET phosphorylation, which correlates with patient outcome after liver surgery. Thus, dysregulated basal MET phosphorylation could be an indicator for the health status of the liver and thereby inform on the risk of a patient to suffer from liver failure after surgery.
... In CHO cells expressing the HA-IRS-1, Ser1101 incubated with insulin and amino acids showed increased phosphorylation of Ser1101 compared with the mutant HA-IRS-1 Ser1101Ala, while tyrosine phosphorylation was higher in the mutant. This discovery signifies a causal factor implicated in the onset of insulin resistance during the satiety processes, relevant to both animal and human physiology [24]. On the other hand, DYRK1A is a kinase that phosphorylates serine and tyrosine residues and also reduces insulin resistance. ...
Insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) are signaling adaptor proteins that participate in canonical pathways, where insulin cascade activation occurs, as well as in non-canonical pathways, in which phosphorylation of substrates is carried out by a diverse array of receptors including integrins, cytokines, steroid hormones, and others. IRS proteins are subject to a spectrum of post-translational modifications essential for their activation, encompassing phosphorylation events in distinct tyrosine, serine, and threonine residues. Tyrosine residue phosphorylation is intricately linked to the activation of the insulin receptor cascade and its interaction with SH2 domains within a spectrum of proteins, including PI3K. Conversely, serine residue phosphorylation assumes a different function, serving to attenuate the effects of insulin. In this review, we have identified over 50 serine residues within IRS-1 that have been reported to undergo phosphorylation orchestrated by a spectrum of kinases, thereby engendering the activation or inhibition of different signaling pathways. Furthermore, we delineate the phosphorylation of over 10 distinct tyrosine residues at IRS-1 or IRS-2 in response to insulin, a process essential for signal transduction and the subsequent activation of PI3K.
... Moreover, PA-dependent GSK3β activation was completely blocked by the mTOR inhibitor rapamycin. Activation of mTORC1 promotes downregulation of insulin signaling through insulin receptor substrate-1 (IRS-1) phosphorylation at serine residues by the ribosomal serine/threonine 6 kinase (S6K), which is an mTOR effector molecule [37][38][39] resulting in GSK3β activation. S199/S202 is a tau site that is also a substrate of PKA, which we found transiently activated 15 min, as well as PKC⍺, after PA exposure. ...
Metabolic diseases derived from an unhealthy lifestyle have been linked with an increased risk for developing cognitive impairment and even Alzheimer’s disease (AD). Although high consumption of saturated fatty acids such as palmitic acid (PA) has been associated with the development of obesity and type II diabetes, the mechanisms connecting elevated neuronal PA levels and increased AD marker expression remain unclear. Among other effects, PA induces insulin resistance, increases intracellular calcium and reactive oxygen species (ROS) production, and reduces the NAD+/NADH ratio, resulting in decreased activity of the deacetylase Sirtuin1 (SIRT1) in neurons. These mechanisms may affect signaling pathways that impact the posttranslational modifications (PTMs) of the tau protein. To analyze the role played by PA in inducing the phosphorylation and acetylation of tau, we examined PTM changes in human tau in differentiated neurons from human neuroblastoma cells. We found changes in the phosphorylation state of several AD-related sites, namely, S199/202 and S214, that were mediated by a mechanism associated with the dysregulated activity of the kinases GSK3β and mTOR. PA also increased the acetylation of residue K280 and elevated total tau level after long exposure time. These findings provide information about the mechanisms by which saturated fatty acids cause tau PTMs that are similar to those observed in association with AD biochemical changes.
Graphical Abstract
... Metabolic function The incidence of metabolic dysfunction, such as insulin resistance, prediabetes, and overt T2D, increases with age and further increases the risk of nearly every age-related condition including heart disease, frailty, cerebrovascular disease, and dementia [23][24][25][26][27]. The nutrient overload model of insulin resistance posits that insulin resistance results, in part, from constitutive activation of mTORC1 leading to S6K1 and Grb10 mediated feedback inhibition on insulin receptor substrate 1/2 (IRS-1/2) [28][29][30]. Consistent with this model, (1) mice lacking S6K1, a downstream target of mTOR, are protected from age and diet-induced insulin resistance [29,31] and (2) rapamycin given intermittently or at a low dose improves metabolic health and insulin sensitivity in mouse models of obesity [32][33][34]. ...
... The nutrient overload model of insulin resistance posits that insulin resistance results, in part, from constitutive activation of mTORC1 leading to S6K1 and Grb10 mediated feedback inhibition on insulin receptor substrate 1/2 (IRS-1/2) [28][29][30]. Consistent with this model, (1) mice lacking S6K1, a downstream target of mTOR, are protected from age and diet-induced insulin resistance [29,31] and (2) rapamycin given intermittently or at a low dose improves metabolic health and insulin sensitivity in mouse models of obesity [32][33][34]. In healthy young men, a single dose of rapamycin (6 mg) attenuated mTORC1 during an amino acid infusion and Age is one of the greatest risk factors for nearly every chronic condition. ...
Treatment with rapamycin, an inhibitor of the mechanistic Target Of Rapamycin Complex One (mTORC1) protein kinase, has been repeatedly demonstrated to extend lifespan and prevent or delay age-related diseases in diverse model systems. Concerns over the risk of potentially serious side effects in humans, including immunosuppression and metabolic disruptions, have cautiously limited the translation of rapamycin and its analogs as a treatment for aging associated conditions. During the last decade, we and others have developed a working model that suggests that while inhibition of mTORC1 promotes healthy aging, many of the negative side effects of rapamycin are associated with “off-target” inhibition of a second mTOR complex, mTORC2. Differences in the kinetics and molecular mechanisms by which rapamycin inhibits mTORC1 and mTORC2 suggest that a therapeutic window for rapamycin could be exploited using intermittent dosing schedules or alternative rapalogs that may enable more selective inhibition of mTORC1. However, the optimal dosing schedules and the long-term efficacy of such interventions in humans are unknown. Here, we highlight ongoing or upcoming clinical trials that will address outstanding questions regarding the safety, pharmacokinetics, pharmacodynamics, and efficacy of rapamycin and rapalogs on several clinically oriented outcomes. Results from these early phase studies will help guide the design of phase 3 clinical trials to determine whether rapamycin can be used safely to inhibit mTORC1 for the treatment and prevention of age-related diseases in humans.
... Inflammatory cytokines, which are elevated in obese individuals, further activate the mTOR pathway, possibly by increasing phosphatidic acid levels in the cell, triggering the production of transcription factors that regulate the synthesis of even more pro-inflammatory cytokines (Conn and Qian 2011). Chronic hyperactivation of mTOR by hyperinsulinaemia can contribute to insulin resistance in obesity by activating S6K, leading to desensitisation of the PI3K/Akt pathway (Um et al. 2004;Khamzina et al. 2005;Tremblay et al. 2007). The mTOR pathway also promotes adipocyte differentiation and hypertrophy, as well as lipogenesis, contributing to fat accumulation (Lamming and Sabatini 2013). ...
The gradual ageing of the world population has been accompanied by a dramatic increase in the prevalence of obesity and metabolic diseases, especially type 2 diabetes. The adipose tissue dysfunction associated with ageing and obesity shares many common physiological features, including increased oxidative stress and inflammation. Understanding the mechanisms responsible for adipose tissue dysfunction in obesity may help elucidate the processes that contribute to the metabolic disturbances that occur with ageing. This, in turn, may help identify therapeutic targets for the treatment of obesity and age-related metabolic disorders. Because oxidative stress plays a critical role in these pathological processes, antioxidant dietary interventions could be of therapeutic value for the prevention and/or treatment of age-related diseases and obesity and their complications. In this chapter, we review the molecular and cellular mechanisms by which obesity predisposes individuals to accelerated ageing. Additionally, we critically review the potential of antioxidant dietary interventions to counteract obesity and ageing.KeywordsAgeingObesityMetabolic syndromeOxidative stressInflammationNutraceuticalsDietary interventions
... mTORC1 acts as a central hub for nutrient signaling, integrating nutritional information as well as hormonal cues to regulate the activation of anabolic processes essential for cell and organismal growth, including protein translation, ribosomal biogenesis, lipogenesis and nucleotide biogenesis; mTORC1 also negatively regulates autophagy [56,57]. mTORC1 is a key downstream effector of insulin signaling and provides feedback inhibition of insulin signaling through its substrates S6K1 and Grb10, thus promoting the degradation of insulin receptor substrate [58][59][60]. As such, sustained activation of mTORC1 by nutrient overload, obesity, and aging has been suggested as an underlying molecular mechanism that drives the development of insulin resistance. ...
Alzheimer's disease (AD) is an age-associated neurodegenerative disease. As the population ages, the increasing prevalence of AD threatens massive healthcare costs in the coming decades. Unfortunately, traditional drug development efforts for AD have proven largely unsuccessful. A geroscience approach to AD suggests that since aging is the main driver of AD, targeting aging itself may be an effective way to prevent or treat AD. Here, we discuss the effectiveness of geroprotective interventions on AD pathology and cognition in the widely utilized triple-transgenic mouse model of AD (3xTg-AD) which develops both β-amyloid and tau pathologies characteristic of human AD, as well as cognitive deficits. We discuss the beneficial impacts of calorie restriction (CR), the gold standard for geroprotective interventions, and the effects of other dietary interventions including protein restriction. We also discuss the promising preclinical results of geroprotective pharmaceuticals, including rapamycin and medications for type 2 diabetes. Though these interventions and treatments have beneficial effects in the 3xTg-AD model, there is no guarantee that they will be as effective in humans, and we discuss the need to examine these interventions in additional animal models as well as the urgent need to test if some of these approaches can be translated from the lab to the bedside for the treatment of humans with AD.
... Obesity-related accumulation of BCAA in plasma has been reported to interfere with the insulin signaling via activation of the mammalian target of rapamycin (mTOR) pathway, precisely the complex mTOR/p70S6K [86][87][88][89], with leucine as the most potent mTOR activator [90]. In the present study, the BWRP-induced decrease in plasma leucine levels (−31.2%) ...
Metabolomics applied to assess the response to a body weight reduction program (BWRP) may generate valuable information concerning the biochemical mechanisms/pathways underlying the BWRP-induced cardiometabolic benefits. The aim of the present study was to establish the BWRP-induced changes in the metabolomic profile that characterizes the obese condition. In particular, a validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) targeted metabolomic approach was used to determine a total of 188 endogenous metabolites in the plasma samples of a cohort of 42 adolescents with obesity (female/male = 32/10; age = 15.94 ± 1.33 year; body mass index standard deviation score (BMI SDS) = 2.96 ± 0.46) who underwent a 3-week BWRP, including hypocaloric diet, physical exercise, nutritional education, and psychological support. The BWRP was capable of significantly improving body composition (e.g., BMI SDS, p < 0.0001), glucometabolic homeostasis (e.g., glucose, p < 0.0001), and cardiovascular function (e.g., diastolic blood pressure, p = 0.016). A total of 64 metabolites were significantly reduced after the intervention (at least p < 0.05), including 53 glycerophospholipids (23 PCs ae, 21 PCs aa, and 9 lysoPCs), 7 amino acids (tyrosine, phenylalanine, arginine, citrulline, tryptophan, glutamic acid, and leucine), the biogenic amine kynurenine, 2 sphingomyelins, and (free) carnitine (C0). On the contrary, three metabolites were significantly increased after the intervention (at least p < 0.05)—in particular, glutamine, trans-4-hydroxyproline, and the octadecenoyl-carnitine (C18:1). In conclusion, when administered to adolescents with obesity, a short-term BWRP is capable of changing the metabolomic profile in the plasma.
... This study found that valine, a branched chain amino acid (BCAA), was enriched in IgAN. BCAAs can activate the mTOR complex 1(mTORC1) pathway (Langenberg and Savage, 2011), which is activated in human skeletal muscle (Tremblay et al., 2007). The former is involved in many anabolic processes, including protein synthesis, cell growth, etc. ...
Background
Immunoglobulin A nephropathy (IgAN) is a highly prevalent glomerular disease. The diagnosis potential of the gut microbiome in IgAN has not been fully evaluated. Gut microbiota, serum metabolites, and clinical phenotype help to further deepen the understanding of IgAN.
Patients and methods
Cohort studies were conducted in healthy controls (HC), patients of IgA nephropathy (IgAN) and non-IgA nephropathy (n_IgAN). We used 16S rRNA to measure bacterial flora and non-targeted analysis methods to measure metabolomics; we then compared the differences in the gut microbiota between each group. The random forest method was used to explore the non-invasive diagnostic value of the gut microbiome in IgAN. We also compared serum metabolites and analyzed their correlation with the gut microbiome.
Results
The richness and diversity of gut microbiota were significantly different among IgAN, n_IgAN and HC patients. Using a random approach, we constructed the diagnosis model and analysed the differentiation between IgAN and n_IgAN based on gut microbiota. The area under the receiver operating characteristic curve for the diagnosis was 0.9899. The metabolic analysis showed that IgAN patients had significant metabolic differences compared with HCs. In IgAN, catechol, l-tryptophan, (1H-Indol-3-yl)-N-methylmethanamine, and pimelic acid were found to be enriched. In the correlation analysis, l-tryptophan, blood urea nitrogen and Eubacterium coprostanoligenes were positively correlated with each other.
Conclusion
Our study demonstrated changes in the gut microbiota and established models for the non-invasive diagnosis of IgAN from HC and n_IgAN. We further demonstrated a close correlation between the gut flora, metabolites, and clinical phenotypes of IgAN. These findings provide further directions and clues in the study of the mechanism of IgAN.
... Excess nutrients and hyperinsulinemia induce mTORC1 and S6K1 overactivation, which further downregulate IRS1 transcription and promote IRS1 degradation. S6K1 kinase phosphorylates IRS1 at serine 1101 (S1101), inhibiting its function and impairing insulin signaling [7]. It has been observed that S6K1 overactivation may disrupt PI3K signaling and inhibits glucose internalization in L6 myocytes and 3T3-L1 adipocytes [8]. ...
... Unregulated inhibition may involve a constitutive activation of S6K1 that could inhibit IRS1 signaling and promote its degradation resulting in IR and the development of metabolic diseases [42]. Several research concerning these mechanisms have identified multiple inhibitory phosphorylation sites in IRS1 that may be involved in the unregulated inhibition of insulin signaling [7,[43][44][45][46]. In addition, it has been suggested that the hyperphosphorylation of S6K1 observed in different IR models may result in the inhibition of both PI3K and Akt kinases [8,9]. ...
... They are further increased after insulin stimulation. Constitutive activation of S6K1 promotes hyperphosphorylation and degradation of IRS1 [7,8]. The configuration settings of phosphorylated sites [46] and other post-translational modifications determine protein function [44]. ...
Background
Insulin resistance (IR) is a condition in which the response of organs to insulin is impaired. IR is an early marker of metabolic dysfunction. However, IR also appears in physiological contexts during critical developmental windows. The molecular mechanisms of physiological IR are largely unknown in both sexes. Sexual dimorphism in insulin sensitivity is observed since early stages of development. We propose that during periods of accelerated growth, such as around weaning, at postnatal day 20 (p20) in rats, the kinase S6K1 is overactivated and induces impairment of insulin signaling in its target organs. This work aimed to characterize IR at p20, determine its underlying mechanisms, and identify whether sexual dimorphism in physiological IR occurs during this stage.
Methods
We determined systemic insulin sensitivity through insulin tolerance tests, glucose tolerance tests, and blood glucose and insulin levels under fasting and fed conditions at p20 and adult male and female Wistar rats. Furthermore, we quantified levels of S6K1 phosphorylated at threonine 389 (T389) (active form) and its target IRS1 phosphorylated at serine 1101 (S1101) (inhibited form). In addition, we assessed insulin signal transduction by measuring levels of Akt phosphorylated at serine 473 (S473) (active form) in white adipose tissue and skeletal muscle through western blot. Finally, we determined the presence and function of GLUT4 in the plasma membrane by measuring the glucose uptake of adipocytes. Results were compared using two-way ANOVA (With age and sex as factors) and one-way ANOVA with post hoc Tukey’s tests or t-student test in each corresponding case. Statistical significance was considered for P values < 0.05.
Results
We found that both male and female p20 rats have elevated levels of glucose and insulin, low systemic insulin sensitivity, and glucose intolerance. We identified sex- and tissue-related differences in the activation of insulin signaling proteins in p20 rats compared to adult rats.
Conclusions
Male and female p20 rats present physiological insulin resistance with differences in the protein activation of insulin signaling. This suggests that S6K1 overactivation and the resulting IRS1 inhibition by phosphorylation at S1101 may modulate to insulin sensitivity in a sex- and tissue-specific manner.
... The association of BCAA with diabetes is thought to be causative 94 . BCAA infusions in rats and humans leads to the development of insulin resistance 95 . This led us to conclude that the observed changes in AA profiles after surgery are likely to be an important factor driving improved insulin resistance. ...
Background
Resolution of type 2 diabetes (T2D) is common following bariatric surgery, particularly Roux-en-Y gastric bypass. However, the underlying mechanisms have not been fully elucidated.
Methods
To address this we compare the integrated serum, urine and faecal metabolic profiles of participants with obesity ± T2D ( n = 80, T2D = 42) with participants who underwent Roux-en-Y gastric bypass or sleeve gastrectomy (pre and 3-months post-surgery; n = 27), taking diet into account. We co-model these data with shotgun metagenomic profiles of the gut microbiota to provide a comprehensive atlas of host-gut microbe responses to bariatric surgery, weight-loss and glycaemic control at the systems level.
Results
Here we show that bariatric surgery reverses several disrupted pathways characteristic of T2D. The differential metabolite set representative of bariatric surgery overlaps with both diabetes (19.3% commonality) and body mass index (18.6% commonality). However, the percentage overlap between diabetes and body mass index is minimal (4.0% commonality), consistent with weight-independent mechanisms of T2D resolution. The gut microbiota is more strongly correlated to body mass index than T2D, although we identify some pathways such as amino acid metabolism that correlate with changes to the gut microbiota and which influence glycaemic control.
Conclusion
We identify multi-omic signatures associated with responses to surgery, body mass index, and glycaemic control. Improved understanding of gut microbiota - host co-metabolism may lead to novel therapies for weight-loss or diabetes. However, further experiments are required to provide mechanistic insight into the role of the gut microbiota in host metabolism and establish proof of causality.
