1. Development of Flow Cytometry Based Methods to Analyze Cellular Insulin Response
By: Adetokunbo Martins
Stony Brook University
Obesity & Metabolism Lab
Department of Human Physiology
University of Oregon

2. Why is Cellular Insulin Response Essential ?
In our lab we are studying obesity and metabolism. This graph to the left displays the trend of obesity among US adults and it has increased in recent decades from about 15 % to an estimation of 45% in the year Insulin resistance is a hallmark metabolic defect in insulin resistance and it increases the risk for developing other diseases such as hypertension and diabetes. Insulin is a hormone that maintains blood glucose levels at about 4 to 7mM and when the blood glucose levels rise this can be very toxic to our bodies. Obesity as many other metabolic diseases introduce many other diseases into our bodies.
Add the legend info where plot comes from
"Evansville Leading America! In Obesity..." Evansville Leading America! In Obesity... N.p., n.d. Web. June 2016.

3. Insulin
Insulin Receptor
Extracellular Surface
Cytoplasm
PI3- K
p110α P
IRS-1
p85α
PIP2
PIP3 P
Defects Within the Insulin Signaling Pathway Can Cause Insulin Resistance
PDK-1
Glycogen Synthesis
Gene activation
AKT
Describe the insulin pathway in detail
Insulin binds to IR
IR auto phosphorylates via its tyrosine kinase activity
IRS-1 recognizes the phosphotryrosines on IR and gets recruited to the membrane. IRS-1 is phosphorylated by IR
IRS1 recruits pI3k to the cell surface
PI3 – K is a heterodimer which consists of regulatory subunits example P85 that I have shown here and catalytic subunits example p110alpha that I have shown here. P110a phosphorylates membrane phosolipid pip2 making pip3
Pip 3 acts as the secondary messenger for all of the down stream signaling events
Pip 3 activates PDK -1 activated phosphorylates AKT
From PI3K to AKT phosphorylation common steps within biochemical pathways so AKT phosphorylation can result in numerous outputs such as glycogen synthesis and gene activation
We care about insulin stimulated glucose uptake so downstream of AKT phosphorylation results in akt substrate as160 being phsophorykated d
Downstream of AS160 phosphorylation Glut 4 gets translocated to the cellular membrane
Glut 4 translocated from inside to the outside but transports glucose from outside to inside
IR gets phosphorylated by tyrosine kinase receptors autophosphorylates as a dimer
Irs-1 recognizes phosphotyrosines on IR gets recruited to the membrane and then IRS is phosphorylates by IR
Regulatory subunits of pi3kinase recognized phostyrosine of Irs 1
Heterodimerized binds with p110a catalytic subunit
PIP 2 and 3 are fatty acids in which get phosphorylated pi3 kinase phosphorylates at the membrane
Receptor tyrosine kinase
Catalytic step – second messenger generation step common to most signaling transduction pathways many PIP 3’s are produced
How the signal gets ampiflied inside the cell
Know the full name
PDK –full name of pip 3 dependednt kinase activated by pip 3 once bound to pip 3 it can do its catalysis and phosphorylate akt
From pi3kinase to akt this is a common core in may different pathways result in different outputs mystery in understanding how signaling transduction works
Concept of signaling nodes can be taken out of this pathway to understand others
AKT is also a kinase can phosprylate other things specifically we care about insulin stimulated glucose ucose uptake
As160 akt substarte 160 kdaltons which gets phosphorylated we do not what happens exactly but we know it requires actine cytoskeleoton
Downstream steps are not well known precise series of events are not well known proceeds through an actin cytoskeleton dependednt pathway
Actin can be in monomers or filaments vesicles get pushed around cells by actin through proteins on the surface that take actin monomers and push the actin filaments
GLUT-4 P
AS16O P

4. Limitations with Current Insulin Response Assays :
We want to assess the function of this pathway in genetically modified mice in obese states
Time and large sample sizes
Dead cells and mixed population so you may not what you are looking at exactly and the cell type that you care about
GOAL: Validation of Flow Cytometry Based Assays to Assess Insulin Function

5. Y Y Flow Cytometry Advantages of Flow Cytometry Based Methods
2 °  Y
1 ° 
Advantages of Flow Cytometry Based Methods
Rapid analysis
Quantitative measurements
Will allow broad response understanding of insulin response in single samples
Can be used on mixed populations of cells
Cells is resuspended ins solution passes thru sample channel one at a time single file interrogated by a laser and info can be retrieved out of that
Fs and ss two parameters detects size and internal complexity of cells
Flow cytometry can detect surface markers in antibodies and uptake in fluorescent dyes
Fluorescent labeled antibodies you can identify surface proteins and intracellular proteins to identify cell populations
Advantages of Flow Cytometry based methods
Rapid analysis
Quantitative measurements
Will allow broad response understanding of insulin response in single samples
Can be used on mixed populations of cells
"Flow FoMD." Flow Cytometry FoMD. N.p., n.d. Web. June 2016.

6. C2C12 Myoblasts as a Model System for Glucose Uptake
Go thru the title – we chose c2c12 as our mice model system
Myoblasts are muscle precursor cells able to divide in cell culture
They can also fully differentiate where they fuse and form into myotubes
Insulin responsive and physiologically relevant cell line
Up to 75% of glucose uptake occurs in skeletal muscle as oppose to adipose tissue physiologically relevant cell line insulin responsive without needing to be differentiated easier to work with for developing methods
Do not have to differentiate
Muscle glucose uptake is the strongest in response to insulin
Makes for rapid experiments and analysis
C2c12 to develop methods
muscle cell that is still able to proliferate
Also capable of proliferating and fusing into myotubes
Differentiated to the point where they are fated to become muscle but they are still able to divide
Gundry RL, et al,. “The mouse C2C12 myoblast cell surface N-linked glycoproteome: identification, glycosite occupancy, and membrane orientation”.

7. Method for Insulin Stimulation
Grow cells to 70% confluency
Serum starve removes FBS from growth media. Deactivation of receptors.
Insulin
Insulin stimulation of 2-NBDG uptake
Look at insulin response in the cells
Process to treat with insulin looking at the outouts
Challeges are if they become to dense they will differentiate into mytuubes
Allows the cells to return to resting state prior to insulin stimulation
Then we have these three downstream outputs that I will be discussing next
+ 100nM Insulin
Detection of AKT phosphorylation
- Insulin
GLUT-4 surface staining

8. Fluorescent Glucose Analog 2-NBDG as a tool for Studying Insulin Stimulated Glucose Uptake+
+ Insulin
2-NBDG
- Insulin +
2-NBDG
Firstly we want to develop a method to one of the endpints of the insulin signaling pathway glucose uptake
]2-nbdg 2 deoxy glucose prevents utilization in glycolysis–it will accumulate in the cell so we will be able to get a lot of it in the cell
Because this is fluorescent it is something we can measure in a flow cytometer
NBD is the actually fluorsent tag that the flow detects

9. Detection of Insulin Stimulated Glucose Uptake+
+ Insulin
2-NBDG
Cell Count
- Insulin +
2-NBDG
Firstly we want to develop a method to one of the endpints of the insulin signaling pathway glucose uptake
]2-nbdg 2 deoxy glucose prevents untilization in glycolysis and fused to that is nbd
Because this is fluorescent it is something we can measure in a flow cytometer
Left peak is low f intensity peak
R is high fluorescent intensity peak
Cells shifted to the r when stimulated with insulin indicating insulin stimulated glucose uptake
2- NBDG Fluorescence Intensity

10. Insulin Stimulated Glucose Fluorescent Analog (2-NBDG) Uptake
Validation that the fluorescence that the flow is detecting is in fact the 2NBDG
Went up in a linear fraction supporting that we had a higher intensity due to the 2nbdg
Nice dose response
Fluorescent intensity
If increasing 2 results in a positive peak then we can conclude that the 2nbdg is working
Because the peak increased proportionally to 2 nbdg evidence
Closure: it looks like 2-nbdg is promising method to use to test insulin stimulated glucose uptake
Fluorescence Intensity

11. Y Measuring Akt Phosphorylation by Flow Cytometry α p-AKT (Thr308)
PI3K
Akt P Y
α p-AKT (Thr308)
AF555 α Rabbit
-pAkt
+ pAkt
Looking at steps along the pathway to determine dysregulation acute measure downstream of PI3 k
We could recognize the AKT through staining it with an antibody that recognized
Cells were fixed and permeablized and fixed again and then stained with primary and secondary anti
Fixing crosslinks the proteins so the insides of the cells does not fall
Fixing it freezes the cell inside with the cellular components
In the future we would want to turn down the voltages so that we can see these cells better but there is definitely a group of well stained cells
α Phospho Thr306 Akt
AF555 α Rabbit

12. Detection of Phosphorylation of Akt by Antibody Staining
By quantifying averaging intensities with the antibodies we have a much higher fluoresce intensities meaning we observed the staining of something
Right one says we says we stained soemthig insulin responsive validating that we can conclude that we are measuring insulin stimulated akt phosphorylation

13. Insulin Stimulated GLUT4 Translocation to the Cellular Membrane
1 ° 
2 ° 
GLUT4
GLUT4
Third method
As previously mention glut 4 is a constitutively active glucose transporter its function is regulated by its localization. Normally glut 4 is sequestered upon insulin stimulation glut4 transports glucose to the cellular membrane
We have an antibody that recognized extracellular component of the transporter

14. GLUT-4 Surface Staining
Treated cells we were able to see an increasing fluorescence when stained
Stained cells were only cells that translocated to the membrane
Permeablized acts as a pseudo concentration dependence
Suprisingly ****
Results did come from one experiment
Not sure need to do more experiments to validate the results

15. Summary & Future Directions
Developed flow cytometry based methods to measure cellular functions downstream of insulin signaling
Glucose uptake
Akt phosphorylation
GLUT4 translocation
Other projects in the lab looks how chaning expression of the regulatory subuinits of pi3kinase affect insulin response and insulin sensitivity in response to high fat diet so these mthods would be used in studying the role of pi3kinase regularoty subunits compostion in insulin signaling
Use methods to assess how regulatory subunits of PI3-K influence insulin sensitivity to high fat diet

16. Acknowledgements Carrie E. McCurdy Lab Byron Hetrick – Mentor
Carrie E. McCurdy – Principal Investigator
William Campodonico
Zach Clayton
SPUR
Peter O’Day – Director
Marilyn Drennan – Coordinator
SBU Faculty
Zachary Katsamanis
David Kahn
SPUR NSF program: NSF REU Site Program in Molecular Biosciences at the University of Oregon: NSF DBI/BIO )

17. Questions ?