Nutrient excess causes insulin resistance, at least in part, because chronic insulin secretion inhibits proximal insulin receptor signals through feedback or heterologous inflammation . Although elevated insulin defends against hyperglycaemia, it can increase hepatic lipogenesis, dyslipidaemia, WAT expansion, and hepatic steatosis. Consequently, elevated circulating and hepatic lipids, including non- esterified fatty acids, diacylglycerol, and ceramides, can activate kinases directly or through inflammatory cascades to further inhibit proximal insulin signalling. Nutrient excess also stimulates mTORC1- mediated feedback mechanisms that can inhibit insulin receptor → IRS1/IRS2 coupling and stability. Regardless, physiological mechanisms that regulate the concentration and function of proximal insulin signal ling components are not understood well enough to guide the development of efficacious and safe treatments.
Transcriptional control of IRS1
Several mechanisms regulate IRS1 transcription, including Wnt/β- catenin signalling. Wnt3A or the constitutively active form of β- catenin increases the IRS1 gene and protein expressions, which decreases during suppression of the Wnt/β- catenin pathway. Moreover, chromatin immunoprecipitation analysis shows that T- cell factor 4 (TCF4), a component of the TCF/LEF lymphoid enhancer factor family, binds to the IRS1 promoter to stabilize Wnt/β- catenin signalling. The TCF/LEF response elements are located between −7000 and −5966 in the mouse IRS1 promoter and transfection by siTCF4 reduces IRS1 mRNA and protein levels in H4IIE cells, suggesting that the β- catenin/TCF4 pathway can regulate IRS1 expression at the transcriptional level. Wnt/β- catenin appears to regulate IRS1 expression in perivenous hepatocytes where lipogenesis predominates. Wnt signalling agonists might modulate glucose homeostasis as a treatment for type 2 diabetes.
IRS1 expression is also regulated by transcriptional repressors, including transcription factor AP2β (AP2β), the p160 family of nuclear receptor coactivators p/CIP (p300/CBP/cointegrator- associated protein), and steroid receptor coactivator- 1 (SRC1). AP2β is expressed in adipose tissue where it pro motes adipocyte hypertrophy, inhibits adiponectin expression, and enhances the expression of inflammatory adipokines such as interleukin 6 (IL- 6) and C- C motif chemokine ligand 2 (MCP1) [134]. AP2β decreases IRS1 mRNA and protein concentration in adipocyte cell lines. Genome- wide association studies reveal AP2β as a candidate gene for the risk of obesity and type 2 diabetes, which might involve negative regulation of IRS1 expression. By contrast, p/CIP and SRC1 serve as transcriptional coactivators for nuclear hormone receptors and certain other transcription factors. IRS1 expression increases on inactivation of p/CIP and SRC1 in mice, which increases glucose uptake and enhanced insulin sensitivity in WAT and skeletal muscle. Finally, muscle- specific TAZ (transcriptional coactivator with PDZ- binding motif) knockout mice display decreased IRS1 expression and insulin resistance. Statins can reduce TAZ levels, which might reveal a mechanism of insulin resistance owing to decreased IRS1 expression; however, effects of statins on protein prenylation might also be involved.
Transcriptional control of IRS2 Unlike IRS1, IRS2 transcription is regulated by nutrient- sensitive factors – including cAMP response element binding protein (CREB) and its coactivator CREB regulated transcription coactivator 2 (CRTC2); FOXO isoforms; hypoxia- inducible factor- 2α (HIF2α) encoded by EPAS1; nuclear factor of activated T cells (NFAT); PGC1A; sterol regulatory element binding protein 1 (SREBP1c); signal transducer and activator of transcription 3 (STAT3); and transcription factor E3 (TFE3). Multiple transcriptional mechanisms induce hepatic IRS2 expression during fasting, including nuclear FOXO that creates a direct feedback loop to augment insulin signalling during fasting and suppress it after a meal through AKT─┤FOXO. Fasting or exercise also induce the CREB•CRTC2 transcriptional complex through glucagon signalling that increases expression of gluconeogenic genes along with IRS2. Moreover, glucagon acting through PGC1A can increase hepatic IRS2 expression while suppressing IRS1, which can finetune gluconeogenesis and hepatic glucose production during the fasting to feeding transition with a minimal effect on lipogenesis. An E- box overlap ping the FOXO site binds basic helix- loop- helix transcription factor E3 (TFE3), which converges with FOXO to promote IRS2 expression; however, these elements also overlap with an SRE that binds sterol regulatory element binding transcription factor 1 (SREBF1), an important transcriptional activator of lipid synthesis. Active hepatic SREBF1 increases during nutrient excess and chronic insulin stimulation, which decreases IRS2 expression to promote insulin resistance while promoting lipogenesis. HIF2α induces transcription of IRS2 in the hypoxic perivenous zone of the liver to restrain gluconeogenesis compared to other liver zones. Thus, IRS2 expression is highly integrated through multiple metabolic sensors to promote insulin sensitivity and metabolic homeostasis.
miRNA- mediated post- transcriptional regulation
MicroRNAs (miRNAs) are short (∼20 nucleotides) non- coding RNA molecules that negatively modulate gene expression through their specific binding within the 3′UTR sequence of messenger RNA (mRNA) to inhibit translation or destabilize the target mRNA. Most of the proximal components of insulin signalling can be regulated in a tissue- specific way by miRNAs. LET7 miRNA inter feres with many proximal components, including type 1 IGF receptor, insulin receptor, IRS2, PIK3IP1, AKT2, TSC1, and RICTOR. LET7 interference can be inhibited by the RNA binding proteins Lin28a and Lin28b, which block production of mature LET7 to increase translation of the insulin signalling components. LET7 associates with gigantism, puberty delay, and glucose homeostasis by modulating insulin → PI3K → mTOR signalling and insulin sensitivity.
Many other miRNAs display specificity against proximal insulin signalling components during metabolic challenge in various tissues. Hepatic miR- 424- 5p, miR- 15b, miR- 195, and miR-96 increase in mouse liver when fed a high- fat diet, which associates with less insulin receptor expression. IRS1 and IRS2 are targeted by miRNAs in multiple peripheral insulin target tissues. miR- 222 can suppress IRS1 in liver and adipose during high- fat diet and sucrose feeding. Resistin upregulates miR- 145 to inhibit IRS1 levels. miR- 29a and miR- 29c suppress IRS1 expression in the muscle of individuals with type 2 diabetes or mice fed high- fat diets. IRS1 can be downregulated in mouse endothelial cells by miR- 126, which dysregulates angiogenesis. Chronic angiotensin- II–induced hypertension can increase the expression of miR- 487b in rat aorta, which suppresses IRS1 expression and promotes hypertension- induced cardiovascular disease and formation of aortic aneurysms owing to the loss of medial smooth muscle.
IRS2 expression is also suppressed by various miRNAs. The miR- 126 targets IRS2 in pancreatic β cells, which can suppress islet growth [159]. Upregulation of miR- 33b reduces hepatic IRS2 → AKT signalling while miR- 135a reduces IRS2 → AKT sig nalling in the muscle of individuals with type 2 diabetes and db/db mice.
Finally, miR- 26b appears to suppress PTEN to promote GLUT4 translocation in adipose and β- cell function during obesity. Other proximal insulin signalling components are also modulated by various miRNAs, including SHIP2 (miR- 205- 5p), PDK1 (miR- 375), AKT (miR- 143─┤ORP8 [oxysterol binding protein related 8]), PP2A (miR- 19b, miR- 429, miR- 29, and miR- 155), and type 1 IGF receptor. Modulating miRNA expression in vivo might be a beneficial strategy to restore insulin sensitivity and treat some forms of type 2 diabetes.
Regulation of IRS signalling by Ser/Thr phosphorylation
IRS1 and IRS2 can be regulated through a complex mechanism involving phosphorylation of more than 50 potential phosphoryl ated Ser/Thr residues (pS/Ts) located in the long unstructured tail (Figure 1). Understanding how phosphorylated Ser/Thr residues regulate insulin signalling is challenging, because so many sites and mechanisms are involved. Heterologous signalling cascades initiated by proinflammatory cytokines or metabolic excess, including tumour necrosis factor α (TNF-α), endothelin- I, angiotensin II, excess nutrients (free fatty acids, ceramides, amino acids, and glucose), or endoplasmic reticulum stress, are implicated in IRS1 and IRS2 phosphorylated Ser/Thr residues, which associate with less insulin- stimulated tyrosine phosphorylation . Most IRS1/2 phosphorylated Ser/Thr residues are stimulated by the PI3K → Akt → mTOR cascade during insulin stimulation. Thus, IRS1/2 phosphorylated Ser/Thr residues are first and fore most a feedback mechanism that develops during chronic insulin stimulation, which can be co- opted by other agonists during metabolic stress to inhibit insulin signalling and dysregulate metabolic homeostasis (Figure 1). Thus, hyperinsulinaemia might be the important physiological mediator of insulin resistance in animals.
Fig1. Schematic diagram of heterologous and feedback regulation of insulin signalling. Various kinases in the insulin signalling cascade mediate feedback inhibition of IRS, including PKB, mTOR, S6K, ERK, AKT, and atypical PKC isoforms [163]. Other kinases activated by heterologous signals, including lipids such as ceramide, are also involved. Serine phosphorylation of IRS1 can recruit CRL7, which can promote ubiquitinylation and degradation of IRS1 through the 26S proteasome. Many proinflammatory cytokines cause insulin resistance through SOCS1 or SOCS3 that targets phosphotyrosine- containing proteins like IRS1 or IRS2 for ubiquitinylation by a BC- containing ubiquitin ligase (E3) and degradation.
Recently, AKT was shown to coordinate negative feedback by phosphorylating IRS1 and IRS2 on several serine residues. AKT- mediated phosphorylation can deplete plasma membrane localized IRS1 and IRS2, which reduces their interaction with the insulin receptor. Reduced membrane- associated IRS protein decreases recruitment and activation of the PI3K, which reduces phosphatidylinositol(3,4,5)- triphosphate (PIP3) synthesis and insulin action. Two AKT- dependent phosphorylation sites in IRS2, mouse S303 and S573 (human S306 and S577), might mediate this negative feedback, but other sites can be involved. These findings establish a novel mechanism by which AKT can feedback to attenuate insulin- stimulated PIP3 production, providing a mechanism to modulate insulin signalling independent of other signal ling pathways.
Mouse S307IRS1 (human S312IRS1) is one of the best- studied pS/T in IRS1, which is often used as barometer of insulin resistance. Insulin can promote pS307Irs1 phosphorylation through the PI3K → AKT → mTORC1 → S6K1 (Figure 1). In mice, free fatty acids promote pS307IRS1, but insulin resistance and hyperinsulinemia are not excluded as the cause. c- Jun N- terminal kinase (JNK1) or mTORC1 activated in obese mice promotes pS307IRS1 and other sites. Although most if not all cell- based investigations support an inhibitory role for pS307IRS1, mouse- based experiments are less convincing. Genetic knock- in to replace S307IRS1 with alanine (A307IRS1) increases fasting insulin and glucose levels while decreasing p110PI3K binding to IRS1 PI3K. During the high- fat diet, A307IRS1 mice exhibit more severe glucose intolerance and higher fasting insulin than control S307Irs1 mice. In mice, pS307Irs1 and other phospho- S/Ts might attenuate the detrimental effects of compensatory hyperinsulinae mia to maintain some relative insulin sensitivity. More work is needed to establish the mechanisms involved.
Regulation of IRS degradation
Proteasome- mediated degradation regulates many biological processes including signal transduction, gene transcription, and cell cycle progression. Proteins targeted for destruction by the 26S proteasome are polyubiquitinylated by various complexes comprising a ubiquitin- activating enzyme (E1), ubiquitin- conjugating enzyme (E2), and ubiquitin- protein ligase (E3). IRS1 and IRS2 can be polyubiquitinylated during chronic inflammatory states, nutrient excess, and hyperinsulinaemia through various tissue- specific mechanisms. The first pathway discovered involves suppressor of cytokine signalling 1 and 3 (SOCS1/3), which binds to IRS1 through its SH2 domain and to a ubiquitin E3 ligase through its SOCS box (Figure 1). Mutations in the SOCS box pre vent ubiquitinylation and degradation of IRS1 or IRS2 [174], and inhibition of SOCS1/3 expression by anti- sense oligonucleotides improves insulin sensitivity in obese and diabetic mice. Cytokines like interferon γ (INFγ) or IL- 6 activate Janus kinases (JAK) promote phosphorylation and dimerization of signal transducer and activator of transcription (STAT) factors, which migrate into the nucleus to induce SOCS1/3 expression (Figure 1). Angiopoietin- like 7 (Angptl7) also increases SOCS3 during obesity. Thus, SOCS- mediated polyubiquitination of IRS1 or IRS2 can promote insulin resistance and glucose intolerance during infection, inflammation, or metabolic stress.
Other mechanisms of IRS degradation are coordinated by pS/T on IRS1 or IRS2. Cullin- RING E3 ubiquitin ligase 7 (CRL7) mediates IRS1 degradation downstream of pS/T generated by the PI3K → AKT → mTORC1 cascade. CRL7 complex contains cullin 7 (CUL7), a molecular scaffold that assembles F- box/ WD repeat- containing protein 8 (FBW8) to recruit phosphorylated substrates, and RING- box protein 1 (Rbx1), associated with an E2 conjugating enzyme (Figure 1). FBW8 apparently binds to IRS1 through pS/T residues generated by the mTORC1 → S6K cascade, including human pS307IRS1, pS312IRS1, and pS527IRS1, but possibly others, to mediate polyubiquitinylation of IRS1 that progresses to degradation (Figure 1). This regulatory mechanism might be engaged during nutrient excess or hyperinsulinaemia, as it depends on chronically hyperactivated mTORC1 → SK6.
Chronic consumption of high- calorie diets upregulates Cbl proto- oncogene B (CBLB), a RING- type E3 ubiquitin ligase that belongs to the casitas B- lineage lymphoma family of proteins. CBL proteins share a conserved NH2- terminal region containing a tyrosine kinase binding domain and a RING- finger domain to facilitate E3 ubiquitin ligase activity. Calorie excess induces carbohydrate- responsive element- binding protein (ChREBP) and SREBP1c, which upregulates myostatin in murine muscle to induce CBLB expression that drives insulin resistance through the polyubiquitinylation and degradation of IRS1.
Regulation by protein and lipid phosphatases
Phosphatases modulate insulin signalling by dephosphorylating key proteins or lipids in the signalling cascade, including PTP1B (tyrosine- protein phosphatase non- receptor type 1, PTPN1), PTPN2 (tyrosine- protein phosphatase non- receptor type 2, TCPTP), protein phosphatase 2A (PP2A), protein phosphatase 1 (PP1), phosphatase and tensin homolog (pTEN), tensin- like C1 domain- containing phosphatase (C1- TEN), and Src homology 2 domain- containing inositol 5′- phosphatase 2 (SHIP2). PTP1B and PTPN2 are related phosphotyrosine phosphatases that dephosphorylate various receptor tyrosine kinases, including the A- loop of the insulin receptor; however, their biological effects can be dis tinct owing to different time courses and tissue expression. PTP1B−/− mice display increased insulin sensitivity, lower circulating insulin concentrations, and decreased pancreatic β- cell mass. Physiologically, both PTP1B and PTPN2 can be inactivated by reactive oxygen species generated during insulin stimulation. In pancreatic β cells, PTP1B attenuates the IRS2 → PI3K → AKT cascade that is important for growth, function, and survival of these cells. Thus, inactivation of PTP1B can maintain β- cell mass in mice lacking IRS2, which prevents the early onset of diabetes. Regardless, without IRS2 even the IRS2−/−•PTP1B−/− mice lose β- cell mass between 8 and 9 months of age, as IRS1 signalling fails to compensate. PTP1B also inhibits leptin signalling (LepRb → JAK2) as it dephosphorylates JAK2. PTPN2 dephosphorylates JAK1/3 but not JAK2. Central nervous system inhibition of PTP1B can protect against obesity, while peripheral inhibition of PTP1B promotes glucose tolerance. Inhibition of PTP1B or TCPTP in the brain, or specific hypothalamic neurons, promotes insulin and leptin signalling and prevents diet- induced obesity, type 2 diabetes, and non- alcoholic fatty liver disease. Regardless, targeting PTP1B and TCPTP for treatment has been problematic due to challenges in targeting the inhibitor to specific tissues; however, intranasal targeting of PTP1B and TCPTP can increase leptin and insulin sensitivity and promote weight loss by repressing feeding and increasing energy expenditure.
pTEN is a potent negative regulator of insulin action and cellular proliferation and is a frequently mutated gene in human cancer [191, 192]. pTEN attenuates insulin signalling by dephosphorylating PI(3,4)P2 and PI(3,4,5)P3 at the 3- position, which reduces the recruitment and activation of PDK1, AKT, and others (Figure 1). pTEN heterozygosity can increase peripheral insulin sensitivity in Irs2−/−•Pten+/− mice and normalizes glucose tolerance, as the small islets in these mice produce enough insulin until death between 10 and 12 months age. These experiments highlight the complex relation between nutrient homeostasis, insulin sensitivity and secretion, and cancer that can emerge in rodents and humans without full pTEN activity.
Phosphatidylinositol- 3,4,5- trisphosphate 5- phosphatase (SHIP2) encoded by the INPPL1 gene attenuates insulin signalling by dephosphorylating the 5′- position of PtdIns(3,4,5) (Figure 1). Several genetic studies link SHIP2 to metabolic disorders, showing that polymorphisms in INPPL1 may contribute to the pathogenesis of the metabolic syndrome, hypertension, and type 2 diabetes. Despite this complexity, SHIP2 inhibition might have therapeutic value. Metformin might increase insulin sensitivity by inhibiting SHIP2, which enhances glucose uptake while protecting renal podocytes from apoptosis in diabetic rodent models.
Phosphoserine- directed phosphatases, including PP2A and PP1, have complex effects depending on substrate targeting. PP2A is a widely expressed pSer/pThr protein phosphatase that forms a heterotrimeric complex with scaffold and regulatory sub units to target various subcellular locations and substrates. PP2A can be a negative regulator of insulin’s metabolic signalling by dephosphorylating and inactivating AKT or dephosphorylating and activating GSK3β. PP2A also targets pS/Ts in IRS1, which stabilizes IRS1 for tyrosine phosphorylation to enhance insulin signalling. PP1 also targets and dephosphorylates IRS1. Phosphoproteomic analysis of L6 skeletal muscle cells reveals the interaction of myosin phosphatase targeting subunit 1 (MYPT1), a targeting subunit of protein phosphatase 1cβ (PP1cβ) with IRS1 [200]. Activation of the MYPT1•PP1 complex by PKA promotes dephosphorylation of serine residues in IRS1 to increase tyrosine phosphorylation and stimulate the PI3K → AKT cascade.
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