- 1.Sample & Assay TechnologiesInsulin ResistanceJesse Liang, Ph.D.
2. Sample & Assay TechnologiesInsulinInsulin is produced by 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 TechnologiesInsulin signalingWhen 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 TechnologiesInsulin resistanceInsulin 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 TechnologiesCauses of insulin resistanceThere are genetic insulin resistance and diet-induced insulin resistance. Obesity is the most common cause of insulin resistance.Overnutritioninflammation oxidative stress lipid metabolism gastrointestinal microbiota (gut microbial influence)Insulin resistance 6. Sample & Assay TechnologiesInflammationAdipose 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: TNF activates JNK and IB Kinase (IKK), which pho IRS-1, hence inhibits insulin receptor signaling. 7. Sample & Assay TechnologiesOxidative stressEvidences: 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 TNF 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 NFB, increase TNF and IL-6 production, and promote macrophage infiltration. 8. Sample & Assay TechnologiesLipotoxicityLipotoxicity: 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 TechnologiesMicrobial contribution to insulin resistanceGnotobiotic (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 TechnologiesThe major contributing or affected sitesInsulin resistance 11. Sample & Assay TechnologiesPPAR- is a major regulator of insulin sensitivityPeroxisome proliferator-activated receptor (PPAR) is the master regulator of adipogenesis, adipocyte differentiation and fat cell gene expression. PPAR is the functioning receptor for the thiazolidinedione (TZD) class of anti-diabetes drugs. These drugs are full classical agonists for this nuclear receptor. PPARFGF1 axis is critical for maintaining metabolic homeostasis and insulin sensitization (Nature, May 2012). PPAR is a crucial molecular orchestrator of visceral adipose tissue Treg cell accumulation, phenotype and function (Nature, June 2012). 12. Sample & Assay TechnologiesPPAR- is a major regulator of insulin sensitivityPPAR- co-activator-1 (PGC1-) 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- at serine 273. This modification of PPAR- 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. PGC1 is a key regulator of mitochondrial content and function. Activities of PGC1- can be modulated by AMPK and SIRT1 (Nature, April 2010). Rb modulates the activity and the expression of Runx2 and PPAR- (Nature, August 2010). 13. Sample & Assay TechnologiesmTOR is another regulator of insulin resistancemTOR stands for the mechanistic target of rapamycin PPAR- 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 TechnologiesA new energy-based conceptA 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 TechnologiesAMPK is a crucial cellular energy sensorAMPK 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 TechnologiesMitochondrial energy metabolismhttp://www.sabiosciences.com/pathwaymagazine/minireview/mitchondrial_energ y_metabolism.php 17. Sample & Assay TechnologiesSirtuins controls acetylation state of histones and transcription factorsOriginally, sirtuins were described as NAD-dependent type III HDACs. The targets of SIRT1 are PGC1, FOXO1, FOXO3, p53, Notch, NF-B, HIF1, 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 TechnologiesRegulation of FOXO by class IIa HDACshttp://www.sabiosciences.com/pathwaymagazine/minireview/metabolichomeostasis.php 19. Sample & Assay TechnologiesmiR-103 and miR-107, miR-143, miR-223, miR-33a and miR-33b, miR-34a, miR-378, let-7miRNAs in insulin resistance 20. Sample & Assay Technologies Gene expression at mRNA level PCR ArraysInsulin 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 Adipogenesis Diabetes Obesity Hypertension Atherosclerosishttp://www.sabiosciences.com/Metabolic_Diseases.php 21. Sample & Assay Technologies Gene expression at mRNA level PCR ArraysInflammatory 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 NFB signaling TGF / BMP signalinghttp://www.sabiosciences.com/Cytokines_Inflammation.php 22. Sample & Assay TechnologiesPCR Array introduction84 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 TechnologiesRT Predictor PCR ArraysHuman Oxidative Stress Toxicity Human Mitochondrial Energy Metabolism Toxicity Human Hypoxia Signaling Pathway Activity Human Unfolded Protein Response Toxicity http://www.sabiosciences.com/pathwayactivity.php 24. Sample & Assay TechnologiesmicroRNA expression microRNA PCR Arrays Liver m