Ch.16 Knockout Mice as a Tool Ch.16 Knockout Mice as a Tool to the Understanding of to the Understanding of

Diabetes MellitusDiabetes Mellitus

2004. 9.14.

CREATION OF KNOCKOUT MICECREATION 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 targetingGene targeting

Insulin Receptor Substrate Knockout ModelsInsulin 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 Normal birth weight Normal glucose homeostasis

IRS-4 KO mice Normal growth Very mild defect in glucose homeostasis

IRS-1/IRS-2 KO mice Embryonic and fetal lethal

IRS-1/IRS-3 KO mice Lipoatrophic diabetes Marked insulin resistance

IRS-1/IRS-4 KO mice Same as IRS-1

⇒ Unique complementary roles of IRS proteins in insulin/IGF-1 signaling cascades

Global Insulin Receptor (IR) Knockout MiceGlobal 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 dec

rease 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 TISSUE-SPECIFIC KNOCKOUT MOUSE MODELSMOUSE 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, eli

minating the DNA that occurs in between .

Tissue-Specific IR KO Mice using Tissue-Specific IR KO Mice using CreCre-loxP-loxP system system

Muscle-Specific Insulin Receptor Muscle-Specific Insulin Receptor Knockout (MIRKO) MiceKnockout (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 skel

etal m. and even a 92% reduction in the heart At birth, no difference in spontaneous activity or exercise capacit

y (~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 The ability of exercise to stimulate glucose uptake A shift of insulin-stimulated glucose uptake and metabolism in

adipose tissue Metabolic syndrome in MIRKO mice

An increased adipose tissue mass Marked hypertriglyceridemia and a modest increase in FFAs

MIRKO mice 1. The utility and value of tissue-specific KO (insulin signaling, glu

cose 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

Fat-Specific Insulin Receptor Fat-Specific Insulin Receptor Knockout (FIRKO) MiceKnockout (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 li

pids, 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 Brown Adipose-Specific Insulin Receptor Knockout (BATIRKO) MiceKnockout (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 Liver-Specific Insulin Receptor Knockout (LIRKO) MiceKnockout (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)

Fed serum insulin levels

Fasting for 16 hrs

→ 2g/kg B.wt of glucose

2 mo old age / male

Metabolic phenotype of hepatic insulin resistance 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

Small livers (50-70% of normal size) 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 insulin-sensitizing agents Neither rosiglitazone nor metformin altered glucose tolerance o

r insulin tolerance Tx. with rosiglitazone reduced LDL-cholesterol levels

Isolated liver insulin resistance Is sufficient to cause severe defects in glucose and lipid ho

meostasis, 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 Liver-Defects in Muscle-, Fat-, and Liver-Specific Insulin Receptor Knockout MiceSpecific Insulin Receptor Knockout Mice

Vascular Endothelial Cell-Specific Insulin Vascular Endothelial Cell-Specific Insulin Receptor Knockout (VENIRKO) MiceReceptor 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 h

omeostasis 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 rol

e in maintaining vascular tone and regulation of insulin sensitivity to dietary salt intake

Pancreatic Pancreatic ββ-Cell-Specific Insulin Recept-Cell-Specific Insulin Receptor Knockout (or Knockout (ββIRKO) MiceIRKO) 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 respons

e 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 respo

nse 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 β-

cell proliferation and insulin secretion

Neuron-Specific Insulin Receptor Neuron-Specific Insulin Receptor Knockout (NIRKO) MiceKnockout (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 as

sociated with insulin resistance and hypertriglyceridemia Hypogonadotropic hypogonadism, associated with impaired m

aturation 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 ore

xin expression Inhibition of insulin receptor affects signaling through melanoc

ortin pathway

DIFFERENT SITES OF INSULIN RESISTANCE PRODUCE DIFFERENT PHENOTYPES

Muscle-Specific Glucose Transporter 4 Muscle-Specific Glucose Transporter 4 Knockout (MG4KO) MiceKnockout (MG4KO) Mice

Glucose transporter 4 (GLUT-4) Insulin-sensitive glucose transporter expressed in skeletal m., h

eart, and adipose tissue Mediates glucose transport stimulated by insulin and contractio

n/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 hyperglycem

ia, and glucose intolerance No increase in body fat or associated hyperlipidemia

Fat-Specific GLUT-4 Knockout Fat-Specific GLUT-4 Knockout (FG4KO) Mice (FG4KO) Mice (aP2 promoter/enhancer)(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 MODELSPOLYGENIC KNOCKOUT MODELS

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 b

ackground of these mice contribute to or protect them from the development of diabetes

Mice with Compound Defects Mimic Mice with Compound Defects Mimic Human Type 2 DiabetesHuman Type 2 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

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 an

d some rodent models Wide range of insulin resistance

IR/IRS-1 DH KO mice similar human type 2 diabetes 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 Improved Insulin Tolerance in Triple-Heterozygous knockoutsTriple-Heterozygous 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 miceHeterozygosity 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 p85-110 dimer, causing ineffective signalingReduction of p85 : more efficient signaling