2. Sample & Assay Technologies
Insulin
Insulin is produced by β cells of pancreas. Insulin causes cells to absorb
glucose from the blood.
Two types of tissues are most strongly influenced by insulin:
muscle cells (myocytes) and fat cells (adipocytes).
3. Sample & Assay Technologies
Insulin signaling
When insulin knocks, GLUT4 opens the door.
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, Sep. 2012)
4. 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 adipose tissue
fatty acid synthesis and systemic insulin sensitivity (Nature, April 2012).
5. 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
6. Sample & Assay Technologies
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:
TNFα activates JNK and IκB Kinase (IKK), which pho IRS-1, hence inhibits
insulin receptor signaling.
7. 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 glucocortocoid
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 cause 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.
8. Sample & Assay Technologies
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, Feb 2013)
9. Sample & Assay Technologies
Microbial contribution to insulin resistance
Gnotobiotic (germ-free) mice are resistant to obesity induced by high fat diet.
Dysbiosis:
In mouse models of 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.
10. Sample & Assay Technologies
The major contributing or affected sites
Insulin resistance
11. 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 fat cell 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).
12. Sample & Assay Technologies
PPAR-γ is a major regulator of insulin sensitivity
PPAR-γ co-activator-1 α (PGC1-α) expression in muscle stimulates an
increase in expression of FNDC5, a membrane protein that is cleaved
and secreted as a newly identified hormone, irisin. Irisin acts on white
adipose cells in culture and in vivo to stimulate UCP1 expression and a
broad program of brown-fat-like development (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 insulinsensitizing adipokine, adiponectin (Nature, July 2010).
Decreased levels of adiponectin and AdipoR1 in obesity may have
causal roles in mitochondrial dysfunction and insulin resistance. PGC1α 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).
13. Sample & Assay Technologies
mTOR is another 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.
14. Sample & Assay Technologies
A new energy-based 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.
15. 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) 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.
16. Sample & Assay Technologies
Mitochondrial energy metabolism
http://www.sabiosciences.com/pathwaymagazine/minireview/mitchondrial_energ
y_metabolism.php
17. Sample & Assay Technologies
Sirtuins controls acetylation state of histones and transcription factors
Originally, sirtuins were described as NAD-dependent type III
HDACs.
The targets of SIRT1 are PGC1α, FOXO1, FOXO3, p53,
Notch, NF-κB, 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 have been shown to develop 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.
18. Sample & Assay Technologies
Regulation of FOXO by class IIa HDACs
http://www.sabiosciences.com/pathwaymagazine/minireview/metabolichomeostasis.php
19. Sample & Assay Technologies
miR-103 and miR-107,
miR-143,
miR-223,
miR-33a and miR-33b,
miR-34a,
miR-378,
let-7
miRNAs in insulin resistance
21. Sample & Assay Technologies
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Gene expression at mRNA level – PCR Arrays
Inflammatory cytokines and receptors
Chemokines and receptors
Common cytokines
Cytokines and chemokines
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.sabiosciences.com/Cytokines_Inflammation.php
22. 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
23. Sample & Assay Technologies
RT² Predictor PCR Arrays
Human Oxidative Stress Toxicity
Human Mitochondrial Energy Metabolism Toxicity
Human Hypoxia Signaling Pathway Activity
Human Unfolded Protein Response Toxicity
http://www.sabiosciences.com/pathwayactivity.php
25. Sample & Assay Technologies
We also provide services – simply send your samples
• RNA extraction
• DNA extraction
• Illumina chips
• All sorts of PCR Arrays
http://www.sabiosciences.com/servicecore.php
