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Ch.16 Knockout Mice as a Tool to the Understanding of Diabetes Mellitus 2004. 9.14. CREATION OF KNOCKOUT MICE Generation of mice carrying null mutations of the genes encoding proteins in the insulin signaling pathway → determining the role of individual proteins in the molecular mechanism of insulin action the pathogenesis of insulin resistance and diabetes Gene targeting Insulin Receptor Substrate Knockout Models IRS-1 KO mice IGF-1 resistance Growth retardation both pre- & postnatally (40-60% of WT) Insulin resistance (mainly skeletal m.) Secretory defect and reduced insulin synthesis in islets β-cell hyperplasia (IGT but not diabetes) Insulin resistance syndrome (HT, hypertriglyceridemia) IRS-2 KO mice 10% reduction in birth weight Severe insulin resistance (liver) + Defect in β-cell proliferation Diabetes in early life IRS-3 KO mice IRS-4 KO mice Lipoatrophic diabetes Marked insulin resistance IRS-1/IRS-4 KO mice ⇒ Embryonic and fetal lethal IRS-1/IRS-3 KO mice Normal growth Very mild defect in glucose homeostasis IRS-1/IRS-2 KO mice Normal birth weight Normal glucose homeostasis Same as IRS-1 Unique complementary roles of IRS proteins in insulin/IGF-1 signaling cascades Global Insulin Receptor (IR) Knockout Mice Complete lack of IR in human due to mutation of IR gene Severe insulin resistance Severe intrauterine growth retardation Usually mild to moderate diabetes Homozygous IR KO mice Normal intrauterine growth (GR : ~10%) and metabolism After birth, severe insulin resistance Die in 3-7 days in diabetic ketoacidosis Intraperitoneal administration of IGF-1 : prompt and sustained decrease in plasma glucose levels through IGF-1 receptor, not IR ⇒ Insulin receptor is necessary for postnatal fuel homeostasis, but not for embryonic development and metabolic control TISSUE-SPECIFIC KNOCKOUT MOUSE MODELS Global gene KO : a lethal phenotype Conditional KO by using tissue-specific promoters → detailed analysis of the gene in a tissue-specific manner Cre-loxP system Cre bacteriophage recombinase Conditionally inactivate genes in mice in which loxP sites have been introduced flanking some critical element loxP sites 34-bp consensus sequence of bacterial DNA Allow for directional recombination of two segments of DNA, eliminating the DNA that occurs in between . Tissue-Specific IR KO Mice using Cre-loxP system Muscle-Specific Insulin Receptor Knockout (MIRKO) Mice Muscle > 80% of postprandial glucose uptake in humans A site of insulin resistance early in the prediabetic state MIRKO mice (MCK promoter-Cre) Almost complete and specific ablation of IR expression in all skeletal m. and even a 92% reduction in the heart At birth, no difference in spontaneous activity or exercise capacity (~10% reduction in muscle mass) Maintain euglycemia up to at least 20 months of age Normal glucose tolerance tests Rates of insulin-stimulated whole body glucose uptake : ↓45% Insulin-stimulated muscle glucose transport : ↓74% Insulin-stimulated muscle glycogen synthesis : ↓87% Exercise-induced glucose uptake : normal Insulin-stimulated glucose transport in adipose tissue : ↑ x3 Physiologic Consequence of MIRKO mice (4 mo old age) Glucose tolerance tests Glucose uptake into muscle and fat * : p < 0.05 The relatively normal plasma glucose in MIRKO mice Metabolic syndrome in MIRKO mice The ability of exercise to stimulate glucose uptake A shift of insulin-stimulated glucose uptake and metabolism in adipose tissue An increased adipose tissue mass Marked hypertriglyceridemia and a modest increase in FFAs MIRKO mice The utility and value of tissue-specific KO (insulin signaling, glucose homeostasis, and pathogenesis of type 2 diabetes) 2. Some cross-talk between muscle and fat Increase in obesity in people with genetically programmed insulin resistance in muscle Overestimated importance of skeletal m. as a site for glucose disposal Indirect effect of insulin-stimulated glucose uptake into m. ↑Blood flow, locally diffusible mediators (NO, cGMP) 3. Significant role of m. insulin resistance in development of the lipid phenotype of the metabolic syndrome 1. Fat-Specific Insulin Receptor Knockout (FIRKO) Mice Fat tissue Only approximately 10% of glucose uptake Major effects of insulin on adipocytes Promote adipogenesis Stimulate glucose uptake and lipid synthesis Inhibit lipolysis FIRKO mice (white & brown fat KO, aP2 promoter-Cre) Survived well after weaning, and are fertile Lean – low fat mass (↓ 50% in fat pad mass) ↓ 30% in whole-body TG content, with normal circulating lipids, FFAs, and glycerol Resistance to obesity during aging or following induction of a hypothalamic lesion leading to hyperphagia Supernormal glucose tolerance (normal glucose tolerance despite overeating) ⇒ Insulin signaling in adipose tissue Not critical for the maintenance of euglycemia Required for the development and maintenance of normal TG stores in adipocytes Inappropriately high leptin levels for fat mass Heterogeneity in adipocyte cell size (large or small fat cells) Increase in lifespan ⇒ insulin signaling pathways are involved in regulation of longevity and that leanness and not food restriction is the most beneficial factor on the extension of lifespan Brown Adipose-Specific Insulin Receptor Knockout (BATIRKO) Mice Important role of brown adipose tissue Determining peripheral insulin sensitivity Thermal adaptation BATIRKO mice (uncoupling protein-1 (UCP) promoter) Age-dependent loss of brown adipose tissue Deterioration of β-cell function and ↓β-cell mass → hyperglycemia ⇒ The maintenance of an adequate β-cell mass somehow requires brown adipose tissue. Endocrine effect of factors produced in brown adipose tissue (?) Broader metabolic change (?) Liver-Specific Insulin Receptor Knockout (LIRKO) Mice Liver Central role in the control of glucose homeostasis Subject to complex regulation by substrates, insulin, and other hormones Effect of insulin to suppress hepatic glucose output Inhibition of glycolysis initially With prolonged fasting, depends on the ability to inhibit gluconeogenesis Insulin resistance in the liver, and especially the loss of the ability of insulin to suppress hepatic glucose output → closely correlated with fasting hyperglycemia in type 2 diabetes Important role in insulin degradation Clearance of insulin in vivo occurs primarily in the liver Mediated by receptor-dependent mechanism LIRKO mice (albumin promoter/enhancer) 2 mo old age / male Fed serum insulin levels Fasting for 16 hrs → 2g/kg B.wt of glucose Metabolic phenotype of hepatic insulin resistance Small livers (50-70% of normal size) At 2 mo of age Impaired glucose tolerance due to ↑gluconeogenesis Fasting and fed hyperinsulinemia & ↓insulin clearance (Significant ↑islet size to compensate for insulin resistance) Fasting and fed hyperglycemia, insulin resistance At 4 mo of age Fasting hyperglycemia disappeared ↓30-50% Serum TG and FFAs Some morphologic changes with collections of large oval cells Gain less weight after weaning → corrected by 6 wk of age Altered IGF and IGF binding proteins Role of hepatic insulin signaling in the action of insulinsensitizing agents Neither rosiglitazone nor metformin altered glucose tolerance or insulin tolerance Tx. with rosiglitazone reduced LDL-cholesterol levels Isolated liver insulin resistance Is sufficient to cause severe defects in glucose and lipid homeostasis, but not uncontrolled fasting hyperglycemia or diabetes Leads to hyperinsulinemia due to changes in both insulin secretion and insulin clearance TZDs may improve some lipid parameters in the LIRKO mice MTF requires an operating insulin signaling system in the liver Defects in Muscle-, Fat-, and LiverSpecific Insulin Receptor Knockout Mice Vascular Endothelial Cell-Specific Insulin Receptor Knockout (VENIRKO) Mice Insulin receptors on vascular endothelial cells : participate in insulin-regulated glucose homeostasis by Facilitating transcytosis of insulin from the intravascular to extracellular space Promoting vasodilation and enhancing blood flow Generation of signaling mediators VENIRKO mice (Tie2 promoter) ⇒ No major consequences on vascular development or glucose homeostasis under basal conditions ↓30-60% eNOS and ET-1 in endothelial cells, aorta, & heart as assessed by RT-PCR and Northern blotting Lower BP but respond normally to high- and low-salt diet Insulin resistance on the low-salt diet Alters expression of vasoactive mediator and may play a role in maintaining vascular tone and regulation of insulin sensitivity to dietary salt intake Pancreatic β-Cell-Specific Insulin Receptor Knockout (βIRKO) Mice Fasting hyperglycemia in type 1 or 2 diabetes is inevitably associated with some degree of β-cell failure Signaling through receptor tyrosine kinases also participates in control of insulin synthesis and release Complete knockout of Irs1 : defective insulin secretion in response to glucose and amino acids Inactivation of Irs2 : impaired β-cell proliferation BIRKO mice (rat insulin 2 promoter (Rip-Cre) ⇒ 85% reduction in acute first-phase insulin secretion in response to glucose and virtually no response in males Maintained acute insulin release in response to arginine No differences in islet size or in the ratio of β- to non-β-cells at 2 mo of age but no slight increase in islet size and insulin content at 4 mo of age Age-dependent glucose intolerance and some overt diabetes Signaling through receptor tyrosine kinases regulates both βcell proliferation and insulin secretion Neuron-Specific Insulin Receptor Knockout (NIRKO) Mice High expression of insulin and IGF-1 receptors in many brain areas and different cell types (glial & neuronal cells) Glucose metabolism in insulin-independent manner in neurons, unclear role of IR in the brain NIRKO mice (nestin promoter-Cre) Increased food intake and moderate diet-dependent obesity associated with insulin resistance and hypertriglyceridemia Hypogonadotropic hypogonadism, associated with impaired maturation of ovarian follicles in female and reduced spermatogenesis in males, leading to reduced fertility ⇒ Insulin receptors in the brain play a role in the control of appetite and reproduction Through an effect of insulin on neuropeptide Y (NPY) and orexin expression Inhibition of insulin receptor affects signaling through melanocortin pathway DIFFERENT SITES OF INSULIN RESISTANCE PRODUCE DIFFERENT PHENOTYPES Muscle-Specific Glucose Transporter 4 Knockout (MG4KO) Mice Glucose transporter 4 (GLUT-4) Insulin-sensitive glucose transporter expressed in skeletal m., heart, and adipose tissue Mediates glucose transport stimulated by insulin and contraction/exercise MG4KO mice (MCK-Cre) > 90-95% reduction of GLUT-4 protein in skeletal m. and no compensatory increase in GLUT-1 Normal growth curves and ↑weight more slowly after 6 mo ↓92% insulin-stimulated glucose uptake in skeletal m. → ↓whole-body glucose uptake Severe whole-body insulin resistance, fasting hyperglycemia, and glucose intolerance No increase in body fat or associated hyperlipidemia Fat-Specific GLUT-4 Knockout (FG4KO) Mice (aP2 promoter/enhancer) Markedly reduced expression of GLUT-4 in both brown and white adipose tissue but normal expression of GLUT-1 Normal growth curves In vitro ↓ Basal & insulin-stimulated glucose uptake into adipocytes (40/72%) Unaltered basal and insulin-stimulated glucose uptake into skeletal m. In vivo ↓53% Insulin-stimulated whole-body glucose uptake ↓50-67% glycolysis and glycogen synthesis Markedly ↓ insulin-stimulated glucose transport into adipocytes Secondary defects in the in vivo milieu resulting from altered release of specific molecules form fat ↓ 40% Glucose transport into skeletal m. ↓Ability of insulin to suppress hepatic glucose production POLYGENIC KNOCKOUT MODELS Mice with Compound Defects Mimic Human Type 2 Diabetes Type 2 diabetes : polygenic in nature → heterozygous double KO mouse model of IR & IRS-1 IR/IRS-1 double-heterozygous (DH) KO mice Marked insulin resistance ↑ x10 circulating insulin levels ↑ x 5-30 β cell mass on a mixed genetic background Only 50% developed diabetes by 4 to 6 mo of age Several features of interest Delayed onset of diabetes despite genetic nature of insulin resistance Marked synergism between IR defect (<10%) and IRS-1 defect (0%) 50% diabetes until 18 mo f/u → additional gene or genes present in background of these mice contribute to or protect them from the development of diabetes Markedly variable phenotype of IR/IRS-1DH mice depending on the genetic background Due to difference in insulin resistance, rather than β-cell failure F2 intercross between mice on B6& 129Sv background → identify the susceptibility of resistance allele DH male intercross mice IR/IRS-1 DH KO mice similar human type 2 diabetes 60% diabetes at 6 mo of age Wide variation and bimodal distribution in fed blood glucoses Wide range of fed plasma insulin levels Relationship between fed insulin and glucose levels Bell-shaped curve Observed in several studies of human with type 2 diabetes and some rodent models Wide range of insulin resistance Polygenic etiology Genetically programmed insulin resistance Delayed age of onset Biphasic relationship between insulin and glucose levels Genome-wide scan of DH intercross mice (90 polymorphic markers) A locus of ch. 12 : linked to hyperglycemia A locus of ch. 14 : significantly linked to hyperinsulinemia Improved Insulin Tolerance in TripleHeterozygous knockouts Three partial defects in insulin signaling → complexity of polygenic disease IR/IRS-1/IRS-2 triple heterozygote mouse Severely impaired glucose tolerance and a doubling of the incidence of diabetes compared with DH IR/IRS-1/p85 KO Less severely affected than IR/IRS-1 DH mice Heterozygosity for the p85 allele Protect mice from diabetes ↑insulin sensitivity in p85(+/-) and IRS/IRS-1/p85 (+/-) mice P85 > p110 in WT : competition between p85 monomer and p85110 dimer, causing ineffective signaling Reduction of p85 : more efficient signaling
