Who has ever built a cell model for insulin resistance

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anline
anline's picture
Who has ever built a cell model for insulin resistance

I'm trying to build a insulin resistance cell model by treat the cell with high concentrated insulin .But I always fail.Now the result shows that the Insulin resistance group can eat more glucose than the normal group.I'm almost crazy.Seem the cells eat glucose is not depended on the insulin concentration.

Chin Fen Teo
Chin Fen Teo's picture
 Hi Anline,

 Hi Anline,

What kind of cells are you working with? Can you also provide the condition that you used?

Insulin stimulate glucose uptake is indeed a very challenging experiment... One thing that you could use to test the insulin resistant status in high Glc + high insulin condition relatively easy is to look at the Akt phosphoryltion (either T308 or S473) by doing western. Insulin resistance leads to a reduction in Akt phosphorylation. Having done several glucose uptake experiments in my life, I strongly suggest you to confirm your cell model with western before spending more time in getting glucose uptake to work...

Cheers.

anline
anline's picture
I'm using hepG2 to build the

I'm using hepG2 to build the cell model of insulin resistance.
The detail way is subculture the cells in normal way(hepG2 is from ATCC, the medium and subculture procedure are all follow the website of ATCC).After it adhered(about over night), starve the cells from FBS for 24hours,and the add 200nM insulin in the FBS free medium(the control cells will be continued treat with FBS free medium but without insulin).After 24 hours, the cells stay with 200nM insulin group will be considered as insulin resistant group.
For I've no idea how to prove my cells is really have insulin resistance.I just follow the literature(some chinese literature, seems not much English literature mentioned how to build a insulin resistance cell model by using hepG2 and insulin).Because the definition of insulin resistance is the certain amount of insulin can not make the cell eat glucose.So the literature always meansure the glucose uptake rate. The way I measure the glucose uptake rate is using the glucose peroxidase to cause the colour change in the solution.I just purchase the glucose (GO) kits from Sigma.I think the kits works well.Anyway, I just incubate the insulin resistant cells and normal cells with phenol red and FBS free medium for 12hours.And using the kits to detect how much glucose left in the medium.
I hypothesis (And I think if I really have the insulin resistant cells the result should be what I hypothesis)the glucose left in insulin resistant group will be more than the normal group.But seem the results always go to wrong way.Some times will be inverted.

anline
anline's picture
That must be something wrong,

That must be something wrong, right?

Chin Fen Teo
Chin Fen Teo's picture
 Hi anline,

 Hi anline,

I have not tried to induce insulin resistance in herpG2 cells before, thus I can't provide you an established condition. And also, I have never heard of anyone using this GO kit for glucose uptake assay- The gold standard is to measure the level of radioaactive (C14) deoxy-glucose being uptake by the cells in response to insulin. I read the product info on Sigma website on this GO kit, and I honestly don't think it is a vigorous method for evaluating glucose uptake level for insulin resistant research.
 
However, based of what you described, here are my thoughts. 
(1) Is it necessary to serum-starve the cells for 48 hours? In adipocyte models, the routine procedure only serum-starve the cells for 16 to 20 hours (that I am aware of).
(2) You did not mention any acute insulin stimulation, so the question is did you wash off the insulin and culture the cells in insulin free media for couple hours and stimulate with insulin? 
(3) If you really want to measure the insulin stimulate glucose uptake levels, I think you should really go for the standard protocol, as that measurement is directly looking at the glucose uptake level, in contrast to this kit you were using (which is measuring the "leftover" glucose).

One last note, as mentioned in my earlier post, the easiest way (in my opinion) to test the insulin sensitivity of your model is to look at the pAkt levels- It is very easy. You (1) culture cells under normal condition or high Glc+Insulin in the absence of serum for overnight, (2) stimulate with insulin (between 5 min to 20 min- you may check out what is the optimal time for HepG2 cells. My cells usually a good 5 min is enough), (3) harvest whole cell lysate, and (4) western. You can purchase pAkt site-specific antibodies from many companies, I routinely use those from either Millipore or Cell Signaling.

Good luck.

mp241186
mp241186's picture
Hi anline,

Hi anline,

Are you trying to build up a model of HepG2 with chronic insulin resistance or acute insulin resistance. Because your state I think is some what between them.

you can use higher concentration of insulin ( eg., 500nM) for shorter time like 16 h. This will not only reduce your time of study but might give you effective results.
And as suggested by pippuri try to confrim insulin resistance by PAKt levels using WB.

anline
anline's picture
What I'm planning to do is to

What I'm planning to do is to build a insulin resistance cell model by using high concentrated insulin as inducer.I have no idea about the chronic or acute insulin resistance.I just want to make the IR cells have more different than the normal one.
I don't want to test the pAKT by WB, because that will change in even serveral minutes before there is really a insulin resistance exist.You see the pAKT level increasing is not equals to insulin resistance, it just mean the cells responds to insulin.
For glucose utilization assay,I think some scientist also use the glucose oxidase method to test the glucose uptake rate, although I agree that using 14C-glucose is the classic way.
Seems only a few people using hepG2 to build the cell model.I feel so sad

Chin Fen Teo
Chin Fen Teo's picture
 Hi anline,

 Hi anline,

I think I have to clarify on the pAkt in the state of insulin resistance.

Chronic insulin and high glucose treatment leading to insulin resistance in adipocytes is a well-established model first reported by Stephen Marshall and colleagues in the early 90s (you can easily pull out a series of papers by using his name and "insulin resistance" in Pubmed). Yes, there is not that many cases of insulin resistant model for HepG2 cells, because adipocytes and skeletal muscles are the major two players in the field. The conditions to induce insulin resistance in HepG2 cells via hyperglycemia and hyperinsulinemia should not be too far off if you omitted the differentiation steps for adipocytes and skeletal cell lines written in the method section of corresponding references.

As for the phosphorylation of Akt, I may have failed to mention that reduction in acute insulin stimulated Akt phosphorylation is also one of the hallmarks of insulin resistance (Keyword="REDUCTION" in pAkt level). Indeed, the defect in insulin signaling cascade with the outcome in the reduction of pAkt level is one of the link to impinge on the Glut4 transporter in adipocytes and skeletal muscles leading to a decrease in insulin mediated glucose uptake. Thus, measuring the reduction in both insulin-stimulated glucose uptake and insulin-stimulated pAkt are two equally convincing readout for insulin resistance. 

Hope this helps to clear things up a bit. Good luck.

MStekele
MStekele's picture
hi Anline,

hi Anline,

how is your insulin resistance cell model going? I am also planning to set up such a model using the glucose kit from Sigma. Or do you discourage that?

maria

anline
anline's picture
Now, I'm using 500nM insulin

Now, I'm using 500nM insulin to treat the cell for 16hours.I think I do get a significant difference between the normal cell and the insulin resistant cell.But the result is inverted.For what I want to see is the normal group will eat more glucose than the insulin resistant group,but I reply it several times,The insulin resistant group alway eat more than the normal group.
Who have the experience for build a cell model of Inuslin Resistance?Do help me!!!
Anyway the glucose kits works well.

anline
anline's picture
From the literature, I have

From the literature, I have to prove my insulin resistance by measure the glucose uptake rate.Because that is the definition of insulin resistance.And I only can use the glucose kits(the kits works well),because the radiative experiment is not allowed in our lab.

MStekele
MStekele's picture
hi Anline,

hi Anline,

why don't you use high glucose to make your cells insulin resistant, I thought that that is the most applied method, put your cells on 25mM glucose for 24h, than stimulate with insulin and see if the glucose uptake is affected by this. The control cells should take up more glucose, the insulin resistant cells not. You use insulin to make your cells insulin resistant but insulin stimulates the glucose uptake and that is what you are measuring...
another question, do you calculate the glucose uptake per cell, i.e. do you measure the glucose in the medium and count the cells in the wells, or do you assume you have an equal amount of cells? In other words, do you normalize the glucose uptake?

vanshita
vanshita's picture
hi anline, i think u should

hi anline, i think u should starve cells for 48hrs instead of 24hrs but it also depend on how much percentage of serum u r using. slowly increasing the concentration of insulin treatment for longer time. to check the insulin resistance u can incubate cells with radioactive carbon n do the glycogen assay.

windy
windy's picture
Hi, Anline, did u use the TNF

Hi, Anline, did u use the TNF-α pretreatment the HepG2 cells for 24hr before  treating with high con insulin?

pla269
pla269's picture
how to built a cell model for

how to built a cell model for insulin resistance,and how to assess this model,do you have some papers,could you share to me ,my email:pla269@163.com,thanks

Badar
Badar's picture
Hi pippuri,

Hi pippuri,

I want to develop insulin resistance state in adipocytes (3T3-L1) but I am confuse which method is best to make them insulin resistance.I have read many papers in which they have used palmitate to induce insulin resistance.

Would you please suggest  which is the best either palmitate, glucose or any other?Can you provide me with some established condition for treating them.

Waiting for your kind reply.

Many thanks !

Chin Fen Teo
Chin Fen Teo's picture
Hi Badar,

Hi Badar,

There are many ways to induce insulin resistance in adipocytes. The most classical method is, perhaps, chronice high glucose + insulin treatment, since is widely accepted that such condition mimic the hyperglycemia and hyperinsulinemia in diabetic condition.

You can find the condition details in the following paper- PMID:9065437.

However, I want to semphasize that, insulin resistance in L1 adipocytes can be induced by many other inducers and it boils down to which pathway(s) you are trying to study, you may be able to establish an insulin resistant models using those inducers instead of high glc/insulin.

Good luck.

Badar
Badar's picture
 Hi Pippuri,

 Hi Pippuri,
 
Thank you very much for your prompt reply.I will surely read this paper.

Best Regards,

sosa_mehr
sosa_mehr's picture
3T3 L1 adipocyte, is the best

3T3 L1 adipocyte, is the best cell line for inducing insulin resistance?
which inducer is better (FFA, cytokine, insuline,..)?

linyijingchen
linyijingchen's picture
 Hi Anline,

 Hi Anline,
ic.Then,I want to test and verify the experiment,but I always fail.I measure the glucose uptake rate by measure the concentration of the liquid after a certain concentration palmitic on the cell.But I couldnot test the difference with or without palmitic.SO,I want to ask for advice.Thank you very much!

linyijingchen
linyijingchen's picture
I want to build a insulin

I want to build a insulin resistance cell model by palmitic.Then,I want to test and verify the experiment,but I always fail.I measure the glucose uptake rate by measure the concentration of the liquid after a certain concentration palmitic on the cell.But I couldnot test the difference with or without palmitic.SO,I want to ask for advice.Thank you very much

murusa
murusa's picture
I am trying to do glucose

I am trying to do glucose uptake in 3T3-L1 cells but cannot see the clear difference between insulin stimulated cells and non-stimulated. My differentiation protocol is according to ZenBio manual - after starting differentiation with IBMX, Dex, IBMX and insulin I culture cells 48 hours, then change to the high glucose media containing FBS and insulin and culture about 7-10 days. I also found this protocol has been used for glucose uptake assay. But now when I cannot see the difference after insulin stimulation I wonder maybe in such condition I have insulin resistance? is that possible cause I use long time incubation with high glucose and insulin?

maybe I need to change the differentiation protocol and culture cells with insulin only 48 hours following by culture with simple high glucose dmem and FBS??

krince
krince's picture
Hi Anline,

Hi Anline,

Did you manage to get your HepG2 insulin resistance cell model up and running with palmitiate and the glucose assay you mentioned? I am also tyring to get the cell model up but am having difficulty.