During the past 25 years, investigators used genetically manipulated mice to understand the molecular and integrated physiology of insulin action. Proximal insulin signalling steps are similar in all tissues, whereas the downstream effects and heterologous regulation can be tissue specific (Figure 1). For example, in liver, post- prandial Pi3k → Akt inhibits FOXO1, which regulates hundreds of genes including increasing genes for de novo lipogenesis (↑Srebf1, ↑Gck, ↑Fasn) and inhibiting genes for hepatic glucose production (↓Pck1, ↓G6pc). In adipose, Pi3k → Akt activates phosphodiesterase 3β (PDE3β) to reduce cAMP and suppress lipolysis, whereas it inhibits AS160 to stimulate glucose influx (Figure 1). In muscle, Pi3k → Akt inhibits protein degradation while stimulating protein and glycogen synthesis and glucose oxidation (Figure 1). The Akt → mTorc1 branch is common to all tissues and stimulates anabolic pathways, including sterol regulatory element- binding factor 1 (Srebp1c)–mediated hepatic de novo lipogenesis (Figure 9.7). During fasting or insulin resistance, nuclear FoxO1 induces Follistatin (Fst), which provokes WAT insulin resistance and lipolysis to deliver glycerol and non- esterified fatty acids to the liver for hepatic glucose production or re- esterification (Figure 1).
Fig1. Tissue- specific insulin signalling. The insulin receptor is autophosphorylated on multiple tyrosine residues, allowing the docking and activation of multiple signalling molecules, most notably insulin receptor substrates (IRS) proteins. This in turn activates phosphatidylinositol- 3- kinase (PI3K) and AKT to mediate the increases in glucose uptake and metabolism as well as changes in protein and lipid metabolism. While the general pathway is similar in all tissues, the final biological effects are specialized to the roles of insulin in liver (inhibits hepatic glucose production and stimulated de novo lipogenesis), adipose (inhibits lipolysis and stimulates glucose influx), and muscle (inhibits autophagy and stimulates proteins synthesis and glucose influx).
Since regulatory tissue cross- talk complicates the analysis of whole- body gene deletion, Cre- loxP technology or specific viral- mediated expression has been used to investigate the role of IR → IRS → AKT ─┤ FOXO1 signalling in specific tissues. Even tissue- specific gene deletion has limitations, as insulin signal ling is rarely lost completely in common metabolic disease. Moreover, targeted deletions still influence metabolic regulation throughout the organism owing to crosstalk by secreted peptides, metabolites, or neuronal interconnections. Regardless, Cre- loxP technology continues to be valuable to dissect the insulin signalling cascades.
Systemic insulin receptor signalling cascade
Mice lacking the insulin receptor have nearly normal size but less adipose tissue mass at birth, and die a few days after birth with severe hyperglycaemia, pancreatic β- cell failure, and ketoacidosis. Thus, insulin → insulin receptor signalling is essential for postnatal nutrient homeostasis. Insulin receptor–deficient mice can be rescued genetically by transgenic expression of the insulin receptor in the brain, liver, and pancreatic β cells. Unlike mice, humans with rare mutations leadings to a lack of functional insulin receptors display intra- uterine growth retardation, failure to thrive, and hypo glycaemia for weeks until hyperglycaemia ensues. This paradox might involve limited availability of gluconeogenic substrates or extreme hyperinsulinaemia that might activate enough IGF- I receptors to generate some insulin- like actions.
Systemic IRS1 or IRS2 deletion suggests that each substrate can display unique signalling properties in various tissues. The signal ling specificity probably arises from different expression levels, receptor coupling, or feedback regulation. Systemic deletion of IRS1 produces small insulin- resistant mice with nearly normal glucose homeostasis owing to β- cell expansion and life- long compensatory hyperinsulinaemia. These results suggest that IRS1 is the principal mediator of IGF- I–regulated body growth, but is not essential for β- cell growth. By contrast, mice lacking IRS2 display nearly normal body growth and even gain fat mass; however, male IRS2−/− mice develop life- threatening diabetes after 8 weeks of age owing to the progressive loss of β- cell mass that disrupts compensatory hyperinsulinaemia. Female mice develop diabetes more slowly, and many but not all develop severe hyperglycaemia by 6 months. Thus, other mechanisms might promote β- cell growth and survival in females.
Compound deletion of IRS1 and IRS2 appears to be embryonic lethal; however, littermates retaining one allele of IRS1 (IRS1+/−•IRS2−/−) or one allele of IRS2 (IRS1−/−•IRS2+/−) can be born alive [224]. IRS1+/−•IRS2−/− mice develop severe fasting hyperglycaemia and die by four weeks of age as β- cells fail to grow or survive. By contrast, IRS1−/−•IRS2+/− mice have a very small body but have normal or elevated β- cell mass to secrete enough insulin for glucose tolerance. Thus, IRS1 and IRS2 are essential for development and nutrient homeostasis, and some IRS2 is essential for β- cell growth, function, and survival.
Hepatic insulin receptor─┤FOXO1 signalling
The liver is an important site of insulin action that plays a role in systemic glucose and lipid homeostasis. LIRKO (IRL/L•CreAlb; hepatic albumin Cre driver) mice display moderately elevated fasting glucose and severe post- prandial hyperglycaemia and glucose intolerance. LIRKO mice also develop severe hyperinsulinae mia owing to a combination of increased insulin secretion to control hyperglycaemia and reduced hepatic insulin degradation. LIRKO mice display reduced levels of circulating free fatty acids and triglycerides on ordinary chow diets; however, on an atherogenic diet LIRKO mice develop dyslipidaemia that can progress to atherosclerosis. Insulin receptor deletion dysregulates hundreds of hepatic genes, including reduced glucokinase and hexokinase 4 (GCK) and elevated phosphoenolpyruvate carboxyki nase 1 (PCK1), glucose- 6- phosphatase, catalytic subunit (G6PC), and pyruvate kinase (PK1). Chronic hyperinsulinaemia in LIRKO mice exacerbates peripheral insulin resistance, while streptozotocin injections to reduce insulin secretion improve peripheral insulin resistance but fail to suppress hepatic glucose production. Thus, heterologous mechanisms dysregulated by hepatic insulin resistance – including the systemic effects of hyper insulinaemia – might exacerbate hepatic glucose production through indirect mechanisms that promote adipose insulin resistance and delivery of excess glycerol and non- esterified fatty acids to the liver for hepatic glucose production.
For over a decade, we have modelled hepatic insulin resistance by genetic inactivation of hepatic IRS1 and IRS2 in LDKO (Irs1L/L•Irs2L/L•CreAlb) mice. LDKO mice dis play unsuppressed hepatic glucose production, hyperinsulinaemia, glucose intolerance, and diabetes. At first, these results suggested that hepatic IRS1/2 mediates systemic nutrient homeostasis through hepatic insulin signalling; however, white and brown adipose tissue and skeletal muscle are also insulin resistant in LDKO mice, suggesting that dysregulated hepatic metabolism manifests systemically. Remarkably, metabolic health is restored upon hepatic inactivation of FoxO1 in LTKO (LDKO•FoxO1L/L) mice, even though the liver is mechanistically unresponsive to insulin. These findings are confirmed and extended with compound hepatic- specific LIRKO•FoxO1L/L mice or Akt1L/L•Akt2L/L•FoxO1L/L•CreAlb mice.
Genetic disruption of hepatic FOXO1 substantially normalizes the expression of hundreds of dysregulated hepatic genes. Within such mice, FOXO1- dependent gene expression or metabolites generated in hepatocytes and circulating systemically could promote peripheral insulin resistance and the delivery of metabolic intermediates to the liver, including excess glycerol and free fatty acids from adipose tissue. Comprehensive analyses of the LDKO and LTKO mice reveals that Fst, best known for its modulation of transforming growth factor (TGF)- β superfamily members Activin and Myostatin, is a key FOXO1- dependent hepatokine that promotes WAT lipolysis during hepatic insulin resistance. Thus, while failing to restore hepatic insulin signalling per se, disruption of hepatic FOXO1 in LTKO mice and similar models might restore glucose tolerance by normalizing hepatokine secretion.
Insulin signalling and glucose homeostasis in skeletal muscle Skeletal muscle
is a major site for utilization and storage of ingested glucose after a meal. Defective insulin signalling at the level of the insulin receptor and IRS1- associated PI3K is associated with reduced insulin- stimulated muscle glucose storage. Regardless, some but not all aspects of metabolic disease emerge on deletion of skeletal muscle insulin receptor in MIRKO (IRL/L•CreMck; muscle creatine kinase Cre driver) mice. Unexpectedly, hyperglycaemia and hyperinsulinaemia never develop in MIRKO mice, while they do develop mild obesity with elevated circulating free fatty acids and triglycerides. Moreover, deletion of the insulin receptor and type 1 IGF receptor in MIGIRKO mice (IRL/L •IGFIRL/L•CreACTA1; human skeletal muscle actin Cre driver) leads to them displaying more than 60% less muscle mass while glucose and insulin tolerance remain normal, owing, at least in part, to increased basal glucose uptake. Consistent with these results, MDKO (Irs1L/L•Irs2L/L•CreMck) mice also fail to develop hyperglycaemia and glucose intolerance during progressive and fatal skeletal and cardiac muscle autophagy. Akt phosphorylation is completely lost in MDKO mice, suggesting that insulin receptor/type 1 IGF receptor signalling is its major agonist. Isolated skeletal muscles from MDKO mice show elevated basal but absent insulin- stimulated glucose uptake, while glucose metabolism shifts to lactate production, which elevates the AMP/ATP ratio, activating AMP- activated protein kinase (AMPK). Activated AMPK can promote phosphorylation of AS160/ TCB1D4 and TCB1D1 to increase translocation of GLUT4 to the cell surface, which increases glucose uptake during complete insulin resistance.
By contrast to the effect of complete muscle insulin resistance, muscle- specific deletion of GLUT4 dysregulates glucose homeostasis and promotes systemic insulin resistance. MG4KO (Glut4L/L•CreMck) mice develop hyperglycaemia, glucose intolerance, and insulin resistance by 8 weeks of age. These mice also display dysregulated glucose metabolism in adipose and liver. Hyperglycaemia owing to diminished muscle glucose utilization appears to drive heterologous insulin resistance, because normalization of circulating glucose by kidney excretion can restore insulin action in adipose and liver.
Adipose insulin signalling
Insulin signalling promotes adipogenesis, glucose influx, lipid syn thesis, and anti- lipolysis in WAT. WAT is important for the storage of post- prandial glucose as triglyceride and the secretion of signalling factors that regulate appetite and energy homeostasis. WAT mainly expresses IRB without a detectable type 1 IGF receptor; however, both receptors play a role in adipogenesis from progenitor cells. Interpretation of the genetic deletion of adipose insulin receptor is complicated by the Cre- drivers used to generate the various experimental mice, including CreaP2 (FIRKO mice; fatty acid binding protein 4 Cre driver) verses CreAdipo (F- IRKO mice; adiponectin Cre driver). FIRKO mice display variable recombination efficiency in fat depots with some off- target events, whereas F- IRKO mice have more efficient insulin receptor deletion across fat depots with few or no off- target effects. With respect to these caveats, FIRKO mice display beneficial metabolic effects, including systemic insulin sensitivity, normal glucose tolerance, and less fat mass on ordinary chow diets. FIRKO mice also have a longer lifespan, suggesting that leanness and insulin sensitivity are associated with longevity. By contrast, F-IRKO mice display lipodystrophy with hepatomegaly, steatosis, and increased enzymes of de novo lipogenesis. F-IRKO mice develop a full spectrum of non- alcoholic fatty liver disease that can progress to non- alcoholic steatohepatitis, fibrosis, and liver dysplasia, which is more in line with the expected effects of adipose insulin resistance. These phenotypes are exacerbated on compound deletion of the insulin receptor and type 1 IGF receptor by CreAdipo, which promotes lipodystrophy of white and brown adipose tissue accompanied by diabetes, insulin resistance, increased β- cell mass, and ectopic lipid accumulation. Thus, the insulin receptor appears to be essential for the formation and maintenance of WAT, whereas both insulin receptor and type 1 IGF receptor contribute to brown adipose tissue mass development and thermogenesis.
Insulin- regulated GLUT4- mediated glucose transport is rate limiting for glucose influx into white and brown adipose tissue under normal glucose and insulin concentrations. GLUT4 translocation to the plasma membrane is extremely sensitive to insulin in adipose, as submaximal activation of AKT2 can stimulate glucose transport. Before high- fat diet–induced insulin resistance inhibits AKT phosphorylation, GLUT4 translocation is strongly inhibited, suggesting that decreased glucose utilization might reside downstream of proximal insulin receptor and AKT activity. Increased adipose GLUT4 expression can rescue systemic insulin resistance of mice fed the high- fat diet.
Adipose triacylglycerol storage is regulated by circulating factors with opposing effects on lipolysis – including β- adrenergic receptors (βAR1/2/3) and insulin. Activation of βAR1/2/3 stimulates cAMP production to promote PKA- mediated phosphorylation of perilipin 1 (PLIN1), which releases abhydrolase domain containing 5, lysophosphatidic acid acyltransferase (ABHD5) to fully activate patatin like phospholipase domain containing 2 (ATGL/PNPLA2), hydrolysing one fatty acid from triacylglycerol. PKA also phosphorylates hormone- sensitive lipase (HLS) to promote its translocation to PLIN1 to hydrolyse fatty acids from diacylglycerol and produce monoacylglycerol. Insulin inhibits lipolysis to pro mote fatty acid storage as triacylglycerol in adipose lipid drop lets; however, the mechanism is only partly understood, as the deletion of AKT2 only partly impairs the ability of insulin to sup press lipolysis. Regulated lipolysis is critical as it controls the delivery of non- esterified fatty acids and glycerol to the liver, which stimulates hepatic glucose production. Insulin inhibits lipolysis by hydrolysing cAMP through an uncertain mechanism. Originally, AKT was thought to activate PDE3β by phosphorylation; however, the direct phosphorylation of PDE3B by AKT might not be required. Instead, AKT phosphorylates α/β hydrolase domain- containing protein 15 (ABHD15), which binds and stabilizes PDE3B for the anti- lipolytic action of insulin.
IRS2 as a gateway to β- cell function
The capacity of pancreatic β cells to maintain glucose homeostasis during chronic physiological and immunological stress is important for cellular and metabolic homeostasis.
Pancreatic β cells have a special place in nutrient homeostasis as the unique source of insulin that also require insulin signalling for growth, function, and survival (Figure 2). Since β cells are always exposed to insulin and IGFs, the proximal signalling cascade appears to be regulated through multifactor transcriptional control of IRS2 – including FOXO1/3, NFAT, and CREB•CRTC2 (Figure 2). In β cells, FOXO can account for as much as 80% of IRS2 expression. Since the IRS2 → PI3K → AKT cascade phosphorylates and inhibits FOXO, insulin or IGF- I has inhibitory effects on FOXO- mediated transcription of IRS2. Since β- cell mass and function must be protected or expanded during chronic nutrient excess, other mechanisms, especially glucose- stimulated Ca2+ influx and cAMP production, promote IRS2 expression to avoid progressive β- cell failure (Figure 2). In addition to its immediate role in insulin secretion, glucose → Ca2+ release activates calcineurin, which dephosphorylates NFAT to facilitate its entry into the nucleus, where it induces expression of IRS2 and other genes (Figure 2). Glucose, glucagon- like peptide 1 (GLP- 1), and other G- protein–coupled receptor (GPCR) agonists also increase cAMP in β cells, which has many important effects, including the activation of CREB•CRTC2 that promotes IRS2 transcription. Through this mechanism, the role of insulin or IGF- I to regulate signalling in β cells is replaced by indirect control through glucose, incretins, or neuronal signals, the physiologically relevant regulators of pancreatic β- cell function (Figure 2). IRS2 expression through these mechanisms maintains PDX1 action, which is essential for β- cell growth, function, and survival. Compounds that augment IRS2 expression might provide treatments for β- cell failure during insulin resistance and the progression of type 2 diabetes.
Fig2. The integrative role of IRS2 signalling in pancreatic β- cell function. The diagram shows the relation between the IRS2 branch of the insulin signalling pathway and upstream and downstream mechanisms regulating β- cell growth and function. Since the insulin receptor and type 1 IGF receptor are constitutively active in β cells, activation of GLP1 → cAMP → PKA → CREB, glucose → Ca2+ → CRTC2, and calcineurin → NFAT induces IRS2 expression to stimulate the PI3K → AKT cascade, which places β- cell growth, function, and survival under the control of glucose and natural or synthetic incretins.
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