Sample & Assay Technologies

Insulin Resistance

Contact Technical Support

North America:• SAB_Support@QIAGEN.com• techservice-na@qiagen.com

Phone: 1-800-362-7737

International customers:BRCsupport@qiagen.com

Webinar related questions: Qiawebinars@qiagen.com

Welcome!

Sample & Assay Technologies Legal Disclaimer

� QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

� For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.

2

Sample & Assay Technologies Outline

I am going to talk about 4 mechanisms and 5 molecule families.

Sample & Assay Technologies Insulin

Insulin is produced by beta cells of pancreas. Insulin causes cells to absorb glucose from the blood.

Two types of cells are most strongly influenced by insulin: muscle cells (myocytes) and fat cells (adipocytes).

Alpha cells synthesize glucagon – blood glucose level

Beta cells produce insulin – blood glucose level

Sample & Assay Technologies Insulin signaling

When insulin knocks, GLUT4 opens the door (insulin stimulates glucose uptake).Measures of insulin resistance were lower in the patients with a PTEN mutation than in controls.The patients' insulin sensitivity could be explained by the presence of enhanced insulin signalingthrough the PI3K-AKT pathway, as evidenced by increased AKT phosphorylation(New Engl J Med, September 2012).

http://www.qiagen.com/products/genes%20and%20pathways/Pathway%20Details?pwid=253

Sample & Assay Technologies Insulin resistance

Insulin resistance (insulin insensitivity, glucose intolerance) means that cells are less responsive to insulin.

Insulin stimulates glucose uptake in adipose tissue through the GLUT4 glucose transporter. GLUT4 expression or function regulates insulin sensitivity.

Carbohydrate-responsive element-binding protein (ChREBP), a downstream target of GLUT4, is a major determinant of systemic insulin sensitivity and adipose tissue fatty acid synthesis (Nature, April 2012).

Sample & Assay TechnologiesCauses of insulin resistance

There are genetic insulin resistance and diet-induced insulin resistance.

Obesity is the most common cause of insulin resistance.

Overnutrition

inflammation oxidative stress lipid metabolism gastrointestinal microbiota(gut microbial influence)

Insulin resistance

Sample & Assay Technologies Obesity and Inflammation

Adipose tissue inflammation

• Invasion of visceral adipose tissue by pro-inflammatory macrophages is considered to be a key event driving adipose-tissue inflammation.

• Visceral adipose tissue has a much greater negative metabolic effect than subcutaneous adipose tissue.

Liver inflammation

Hypothalamic inflammation

Central nervous system inflammation

Mechanisms:Infiltrating macrophages secret TNFα. TNFα activates JNK and IκB Kinase (IKK), which phophorylate IRS-1, hence inhibits insulin receptor signaling.

Sample & Assay Technologies Oxidative stress

Evidences:

Several cell culture models have shown that treatment of insulin-responsive cell lines with H2O2 causes a significant decrease in insulin sensitivity.

Induction of insulin resistance in cell culture with TNFα or glucocorticoid treatment causes a significant induction of ROS. Insulin resistance in these models can be prevented by treatment with different antioxidant compounds.

Mechanisms:

1. Oxidative stress stimulates stress signaling such as JNK, that in turn causes inhibition of insulin signaling (mice without JNK1-signaling do not develop insulin resistance under dietary conditions that normally produce it).

2. ROS trigger the expression of MCP-1, activate NFκB, increase TNFα and IL-6 production, and promote macrophage infiltration (in other words, ROSpromote inflammation).

Sample & Assay Technologies Obesity and LipotoxicityLipotoxicity:Increased levels of circulating fatty acids and/or lipid accumulation inside thecells can lead to insulin resistance.

The origins and drivers of insulin resistance (Cell, February 2013)

Sample & Assay Technologies Microbial contribution to insulin resistance

Gnotobiotic (germ-free) mice are resistant to obesity induced by high fat diet.

Dysbiosis:Dysbiosis refers to a condition with microbial imbalances on or within the body.In obesity, the composition of the microbiota is altered.

A dysbiotic microbiota contributes to insulin resistance by increased energy harvest and the direct effect of altered bacterial metabolite production (e.g.,short-chain fatty acids and bile acid derivatives) on the liver and adipose tissue. In addition, gastrointestinal permeability caused by either intestinal inflammation or intestinal immune dysfunction results in penetration of bacteria or bacterial products (e.g., LPS, DNA), which can drive systemic inflammation.

US Food and Drug Administration (FDA) in April, 2013 declared thatfecal microbiota transplants (FMT) will be considered and regulated as biologic drugs.

QIAGEN offers the largest qPCR assay collection for microbiome researchhttp://www.qiagen.com/landing-pages/sample-technologies/microbiome-metagenomics?sc_mode=normal

Sample & Assay Technologies The major contributing or affected sites

Insulin resistance

Sample & Assay Technologies PPAR-γ is a major regulator of insulin sensitivity

Peroxisome proliferator-activated receptor (PPARγ) is the master regulator of adipogenesis, adipocyte differentiation and adipocyte gene expression.

PPARγ is the functioning receptor for the thiazolidinedione (TZD) class of anti-diabetes drugs. These drugs are full classical agonists for this nuclear receptor.

PPARγ–FGF1 axis is critical for maintaining metabolic homeostasis and insulin sensitization (Nature, May 2012).

PPARγ is a crucial molecular orchestrator of visceral adipose tissue Treg cell accumulation, phenotype and function (Nature, June 2012).

Sample & Assay Technologies PPAR-γ is a major regulator of insulin sensitivity

FNDC5 / irisin, liberated by muscle tissue in response to exercise, drives brown fat development of white fat. PPAR-γ co-activator-1 α(PGC1-α) expression in muscle controls irisin (Nature, Jan 2012).

Obesity induced in mice by high-fat feeding activates Cdk5 in adipose tissues. This results in phosphorylation of PPAR-γ at serine 273. This modification of PPAR-γ does not alter its adipogenic capacity, but leads to dysregulation of a large number of genes whose expression is altered in obesity, including a reduction in the expression of the insulin-sensitizing adipokine, adiponectin (Nature, July 2010).

Decreased levels of adiponectin and AdipoR1 in obesity may havecausal roles in mitochondrial dysfunction and insulin resistance. PGC1-α is a key regulator of mitochondrial content and function. Activities of PGC1-α can be modulated by AMPK and SIRT1 (Nature, April 2010).

Rb modulates the activity and the expression of Runx2 and PPAR-γ (Nature, August 2010).

Sample & Assay Technologies mTOR is another major regulator of insulin resistance

mTOR stands for “the mechanistic target of rapamycin”

PPAR-γ and mTOR are 2 major regulators of insulin sensitivity.

mTORC1 and mTORC2 play distinct roles:mTORC1 inhibits insulin signaling through its substrate S6K1;mTORC2 increases insulin signaling through phosphorylation of AKT.

mTOR signaling in growth control and disease (Cell, April 2012).

Sample & Assay Technologies A new concept

A new energy-based concept of insulin resistance, in which insulin resistance is a result of energy surplus in cells, starts to reinterpret literature for a unifying mechanism of insulin resistance. The energy surplus signal is mediated by ATP and sensed by AMPK signaling pathway. Decreasing ATP level by suppression of production or stimulation of utilization is a promising approach in the treatment of insulin resistance.

Sample & Assay Technologies AMPK is a crucial cellular energy sensor

AMPK stands for “AMP-activated protein kinase”.

AMPK monitors cellular energy status by sensing increases in the ratios of AMP/ATP and ADP/ATP.

AMPK is activated by low energy status (increased AMP/ADP: ATP ratio) suchas during exercise, and regulates metabolic process and energy homeostasis by switching off ATP consuming pathways (fatty acid and cholesterol synthesis) and switching on ATP generating processes (glucose uptake and fatty acid oxidation).

Activation of AMPK promotes glucose uptake, fatty acid oxidation, mitochondrial biogenesis, and insulin sensitivity.

Please try RT2 Profiler AMPK Signaling PCR Array

Sample & Assay Technologies Mitochondrial energy metabolism – homework 1

http://www.sabiosciences.com/pathwaymagazine/minireview/mitchondrial_energy_metabolism.php

Sample & Assay TechnologiesSirtuins control acetylation state of histones, transcription factors and specific proteins in cytoplasm and mitochondria

Originally, sirtuins were described as NAD-dependent type III HDACs. In mammals, there are SIRT1 - SIRT7.The targets of SIRT1 are PGC1α, FOXO1, FOXO3, p53, Notch, NFκB, HIF1α, LXR, FXR, SREBP1c and more.

Sirtuin activation prevents diet-induced obesity.

SIRT1 activation protects from diet-induced and genetic insulin resistance in mice. Mice lacking SIRT1 specifically in the liver developed insulin resistance.

SIRT3 also has a role in insulin sensitization, as its absence may contribute to the development of insulin resistance in the muscle by increasing ROS production and impairing mitochondrial oxidation. Sirtuins as regulators of metabolism and healthspan (Nature Reviews Molecular Cell Biology, April 2012)

Sample & Assay Technologies Regulation of FOXO by class IIa HDACs – homework 2

http://www.sabiosciences.com/pathwaymagazine/minireview/metabolichomeostasis.php

Sample & Assay Technologies miRNAs in insulin resistance

miR-103 and miR-107,

miR-143,

miR-223,

miR-33a and miR-33b,

miR-34a,

miR-378,

let-7

Sample & Assay Technologies Gene expression at mRNA level – PCR Arrays

• Insulin resistance• Insulin signaling• Glucose metabolism• Fatty acid metabolism• Mitochondrial energy metabolism• Fatty liver• Lipoprotein signaling and cholesterol metabolism• Oxidative stress• Glucocorticoid signaling • Autophagy• PPAR targets• mTOR signaling• AMPK signaling• Adipogenesis• Diabetes• Obesity• Hypertension• Atherosclerosis

http://www.qiagen.com/products/genes%20and%20pathways/complete%20biology%20list/

Sample & Assay Technologies Gene expression at mRNA level – PCR Arrays• Inflammatory cytokines and receptors• Chemokines and receptors• Common cytokines• Cytokines and chemokines• TNF signaling pathway• Antiviral response• Inflammatory response and autoimmunity• Toll-like receptors (TLRs)• Innate and adaptive immune response• Inflammasomes• IL-6/STAT3 signaling• T helper cell differentiation• Th1 and Th2 responses• Th17 response• Interferons and receptors• MAPK signaling• NFκB signaling• TGFβ / BMP signalinghttp://www.qiagen.com/products/catalog/assay-technologies/real-time-pcr-and-rt-pcr-reagents/rt2-profiler-pcr-arrays

Sample & Assay Technologies PCR Array introduction

84 pathway-specific genes of interest

5 housekeeping genes

Genomic DNA contamination control

Reverse transcription controls (RTC) n=3

Positive PCR controls (PPC) n=3

Total RNA extraction cDNA mix with master mix and load the plate

run real-time PCR data analysis

Sample & Assay Technologies miRNA expression – miRNA PCR Arrays

• Liver miFinder

• Diabetes

• Cardiovascular diseases

• Immunopathology

• Inflammatory response and autoimmunity

• Serum and plasma miRNAs

http://www.qiagen.com/products/catalog/assay-technologies/mirna/miscript-mirna-pcr-arrays

Sample & Assay TechnologiesCignal Reporter Arrays or Assays – measure signaling pathway activity in cultured cells

Energy metabolism: how to measure PPAR and FOXO activity?http://www.qiagen.com/products/catalog/assay-technologies/protein-and-cell-assays/cignal-reporter-assay-kits

Nuclear Receptors 10-Pathway Reporter Array- PPAR, RAR, RXR, LXR, ER, AR, PR, GR, VDR, HNF4 http://www.qiagen.com/products/catalog/assay-technologies/protein-and-cell-assays/cignal-finder-reporter-arrays

45-Pathway Reporter ArrayCignal 45-Pathway Reporter Array is a novel cell-based assay for monitoring 45 cell signaling pathways in cultured cells in a single experiment. You can observe phenotypes and quantitate as a dual-luciferase assay.http://www.qiagen.com/products/catalog/assay-technologies/protein-and-cell-assays/cignal-45-pathway-reporter-array

Sample & Assay Technologies We also provide services – simply send your samples

• RNA extraction

• DNA extraction

• Illumina chips

• All kinds of PCR Arrays

http://www.qiagen.com/products/catalog/services/

Sample & Assay Technologies Thank You for Attending!

Contact Technical Support

North America:• SAB_Support@QIAGEN.com• techservice-na@qiagen.com

Phone: 1-800-362-7737

International customers:BRCsupport@qiagen.com

Webinar related questions: Qiawebinars@qiagen.com