The document provides information about insulin resistance and related mechanisms. It discusses how insulin resistance is caused by factors like obesity, inflammation, oxidative stress, and microbial dysbiosis. It outlines key regulators of insulin sensitivity including PPARγ, mTOR, AMPK, sirtuins, and miRNAs. The document promotes QIAGEN products for analyzing gene expression and signaling pathways involved in insulin resistance and related conditions.

1. Sample & Assay Technologies
Insulin Resistance
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3. Sample & Assay Technologies
Outline
I am going to talk about 4 mechanisms and 5 molecule families.

4. 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

5. 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 signaling
through 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

6. 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).

7. Sample & Assay Technologies
Causes 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

8. 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.

9. 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, ROS
promote inflammation).

10. Sample & Assay Technologies
Obesity and Lipotoxicity
Lipotoxicity:
Increased levels of circulating fatty acids and/or lipid accumulation inside the
cells can lead to insulin resistance.
The origins and drivers of insulin resistance (Cell, February 2013)

11. 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 that
fecal microbiota transplants (FMT) will be considered and regulated as biologic
drugs.
QIAGEN offers the largest qPCR assay collection for microbiome research
http://www.qiagen.com/landing-pages/sample-technologies/microbiome-metagenomics?sc_mode=normal

12. Sample & Assay Technologies The major contributing or affected sites
Insulin resistance

13. 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).

14. 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 have
causal 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).

15. 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).

16. 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.

17. 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) such
as 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

18. Sample & Assay Technologies
Mitochondrial energy metabolism – homework 1
http://www.sabiosciences.com/pathwaymagazine/minireview/mitchondrial_energy
_metabolism.php

19. Sample & Assay Technologies
Sirtuins 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)

20. Sample & Assay Technologies Regulation of FOXO by class IIa HDACs – homework 2
http://www.sabiosciences.com/pathwaymagazine/minireview/metabolichomeostasis.
php

21. 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

22. 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/

23. 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 signaling
http://www.qiagen.com/products/catalog/assay-technologies/real-time-pcr-and-rt-pcr-reagents/rt2-profiler-pcr-arrays

24. 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

25. 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

26. Sample & Assay Technologies
Cignal 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 Array
Cignal 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

27. 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/

28. 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