Leptin - Science topic

You have more than one option here.

1- You have to look at the cells under the microscope to see if there are any dead cells, there may be something wrong with the counter.

2- For the positive control, it is very strange actually but you have to mention it and search for its effect on the HCT116 cell line in the literature

3- The concentrations you are using with the positive control is very low to cause cell death.

4- if you have to take care also for the confluency of cells in both the control and the cells to be treated, they should be the same before treatment.

Hope to hear about the positive control you are using

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Dear Yasuhiro,

Thanks again for your reply. I understand better. I planned to do subgroup analysis by BMI. I hope this would help to better understand the associations.

Kind regards,

Magda

Physiological and behavioral factors, gastrointestinal factors, disease-associated factors, and environmental factors that initiate the hunger center can affect the feeling of hunger.

Leptin is a small peptide hormone (16-kDa protein) that is mainly, but not exclusively, produced in adipose tissue. The circulating leptin concentration therefore directly reflects the amount of body fat.

is many evidences supporting the statement that changes in insulin secretion and glucose metabolism are the major mediators of leptin production by adipose tissue .

but not article this hormone affect on Hematological Parameters

Plasma dissolution and centrifugation to obtain the serum, then measurement of leptin hormone by ELISA kit

Emilie Doeraene I used the ELISA Kit (Solarbio) for the same purpose (measuring blood leptin levels) and I used it as such without any dilution. In your case, Please check the protocol and it will be specified in the Kit manual.

Dear Rachel!

Please You look ar the article:

Table 1

Summary of animal models of obesity described in this chapter.

Thanks for the suggestions from Tahir and Marie. Both of what you said is good points to the experiment. While designing the experiment, I have considered some factors, such as leptin resistance, injection time, age et.al.

Finally I fixed this problem by using the dissolving buffer recommended by the company and extending the dissolving time up to 30min. This time, even the moderate dosage with 2mg/kg could reduce animals' body weight remarkably.

Sai Shilpa Kommaraju - by background, I mean the same sample background, how do you know you don't get some kind of interference from the sample? another question - are all of your readings in the linear range?

Yes. leptin work against weight loss

This manuscript might help you

Not sure about RAT (renin angiotension?) but when measuring HbA1c in animals, note that the human methods that rely on beta N terminal glycation, including charge based alterations chromotographic mobility may not be appropriate. Immunoassays based on human haemoglobin glycation might also be suspect. Methods that generally measure overall haemoglobin glycation - ie affinity methods eg Beckman & Tosoh may be most appropriate.

Sorry I just bumped into your question. We have a long experience doing both of these assays in our lab, measuring them in plasma and reading the plates (384-well) with the FluoStar Omega reader. What is it that you need to know? The type of sample, the assay to use or the parametres to set in the reader?

Adiponectin, resistin and visfatin

ELISA is the best way

herewith a R&D ELISA kit for Human leptin

You can check

Dear Faek Ali,

Maybe the following papers will help you on the subject:

Ruhl CE, Everhart JE, Ding J, Goodpaster BH, Kanaya AM, Simonsick EM, Tylavsky FA, Harris TB; Health, Aging, and Body Composition Study. Serum leptin concentrations and body adipose measures in older black and white adults. Am J Clin Nutr 2004;80(3):576-83. https://academic.oup.com/ajcn/article/80/3/576/4690533

Ruhl CE, Harris TB, Ding J, Goodpaster BH, Kanaya AM, Kritchevsky SB, Simonsick EM, Tylavsky FA, Everhart JE. Body mass index and serum leptin concentration independently estimate percentage body fat in older adults. Am J Clin Nutr 2007;85(4):1121-6. https://academic.oup.com/ajcn/article/85/4/1121/4648768

Nazish Rafique, Mohammad Nasir Afzal. Relationship of serum leptin levels with body mass index and gender. Rawal Medical Journal 2009;34(2):1-11. https://www.ejmanager.com/mnstemps/27/27-1302359611.pdf?t=1552645414

Shah NR, Braverman ER. Measuring adiposity in patients: the utility of body mass index (BMI), percent body fat, and leptin. PLoS One 2012;7(4):e33308. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3317663/pdf/pone.0033308.pdf

“Leptin values are appromiately 2.5 times higher in women than men per unit BMI.” http://www.kumc.edu/Documents/siddrc/dma/immuno_PDF/leptin%20human.pdf

Best wishes from Germany,

Martin

You implied to express the measured OD by the given concentration. If you have the OD given and you want to know the concentration, you would have to find the inverse function. There might be some smart trick in drc, but I solved the problem "manually" with a function that searches the x-values (conc) at which a given y-value (od) is obtained:

inverse <- function(fit, y, interval="prediction") {

rootFn <- function(value, which)

predict(fit, new=data.frame(value), interval=interval)[which]-y

mean <- uniroot(rootFn, interval = range(fit$dataList$dose), which = 1)$root

if (is.na(interval)) return(mean)

c(

Prediction = mean,

Lower = uniroot(rootFn, interval = range(fit$dataList$dose), which = 3)$root,

Upper = uniroot(rootFn, interval = range(fit$dataList$dose), which = 2)$root

)

}

inverse(fit, 2)

## Prediction Lower Upper ## 601.1108 583.4868 619.1450

So if the measured OD is 2.0, the predicted conc. is with 95% confidence between 583 to 619 (the best guess is 601).

Simpler but actually not really correct is to fit the inverse function:

invfit <- drm(conc~od, fct=LL.4()) predict(invfit, new=data.frame(od=2), interval = "prediction") ## Prediction Lower Upper ## 620.0661 568.6277 671.5045

You see that the results are similar, but not identical.

---

The scatterplot shows the given data, OD on the y-axis and conc. on the x-axis. The solid red line ist the fitted model curve, and the dotted lines are the limits of the 95% prediction interval. Not sure what your problem is here.

Leptin resistance is the more appropriate. Its generally defined as the failure of endogenous or exogenous leptin to promote anticipated salutary metabolic outcomes in states of over-nutrition or obesity, although the hormone's inability to promote desired responses in specific situations results from multiple molecular, neural, behavioral, and environmental mechanisms.

Please check

Please check

Is any relation to insulin plasma concentration

Hello, see links below and attached info all about leptin.

Good luck!

Hi Yanuar

I've found lots of old papers in rats. Handling rats is always easier, their brain and bodies are x10 times larger, everything is easier with them. But since genetically modified animals are usually mice, they have become the species-of-use in the last two decades.

Optogenetics and pharmacogenetics can be done in rats as well, provided you get viral vectors designed for rats, which is not so difficult.

Both species are similar but not identical. Both are rodents, and good models for studying interesting issues for humans, I don't find advantages in mice or rats relative to their similarity to humans.

Good luck

Fernando

Since the discovery of leptin there is a considerable amount of research has focused on leptin as a central regulator of body weight. In the animal model, research has demonstrated leptin action through hypothalamic centres altering both satiety and energy expenditure. In contrast to animal studies, it is unlikely that leptin functioning in the human system exerts such a profound role in body weight regulation. Human studies suggest that leptin levels are strongly correlated with both percentage fat mass and body mass index, in accordance with the proposed 'lipostatic theory'. It has been demonstrated that polyunsaturated fatty acid (PUFA) intake influences adipose tissue expression of leptin, and of several lipogenic enzymes and transcription factors. In addition, leptin stimulates triglyceride depletion in white adipose tissue without increasing free fatty acid release, thus favouring fatty acids versus glucose as a fuel source. Recent studies also suggest that the reduction in adipose hypertrophy observed with n-3 PUFA-containing fish oil feeding might involve a leptin-specific process. A large amount of evidence supports direct functioning of leptin in peripheral lipid metabolism in vivo and in vitro. It is possible that PUFAs will maintain an efficient level of circulating leptin, thus preventing leptin insensitivity and weight gain.

1. Sáinz N, Rodríguez A, Catalán V, Becerril S, Ramírez B, Lancha A, et al. (2012) Leptin Reduces the Expression and Increases the Phosphorylation of the Negative Regulators of GLUT4 Traffic TBC1D1 and TBC1D4 in Muscle of ob/ob Mice. PLoS ONE 7(1): e29389. https://doi.org/10.1371/journal.pone.0029389

2. Cammisotto P, Bendayan M. A review on gastric leptin: the exocrine secretion of a gastric hormone. Anatomy & Cell Biology. 2012;45(1):1-16. doi:10.5115/acb.2012.45.1.1.

3. Roh C, Thoidis G, Farmer SR, Kandror KV.Identification and characterization of leptin-containing intracellular compartment in rat adipose cells. Am J Physiol Endocrinol Metab. 2000 Oct;279(4):E893-9.

4. Cecilia Roh , Raphael Roduit , Bernard Thorens , Susan Fried , and Konstantin V. Kandror .Lipoprotein lipase and leptin are accumulated in different secretory compartments in rat adipocytes. K. V. Kandror. J. Biol. Chem. published online July 12, 2001

5. Feng Ye, Aung Than, Yanying Zhao, Kian Hong Goh, Peng Chen .Vesicular storage, vesicle trafficking, and secretion of leptin and resistin: the commons, differences and interplays ; Nanyang Technological University;1-35

6. LinglinXie, Cormac P.O'Reilly, Stephen K.Chapes, SilviaMora. Adiponectin and leptin are secreted through distinct trafficking pathways in adipocytes; Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease;Volume 1782, Issue 2, February 2008, Pages 99-108

7. CECILIA ROH, GALINI THOIDIS, STEPHEN R. FARMER, AND KONSTANTIN V. KANDROR ;Identification and characterization of leptin-containing intracellular compartment in rat adipose cells; Am J Physiol Endocrinol Metab;2000

Hello.

I have used this one fro leptin and works fine. You also have adiponectin from the same company. They are a little expensive but they work very well

Leptin:

http://www.emdmillipore.com/US/en/product/Mouse-Leptin-ELISA,MM_NF-EZML-82K?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&bd=1

Adiponectin:

If you have more questions let me know!

Follow this link

kind regards

I think there should be no cost differences, but I would prefer serum estimation than DBS, because of sample retrieval, limited whole blood assay availability, micro sample errors etc.

It's a cell culture

Excellent thanks Russell.

Hi,

I work with mostly pregnant mice (with and w/o 3 weeks of high fat diet), but the leptin levels we detect are very similar to male mice on HFD for longer time periods (~40ng/mL). We also use the Crystal Chem kits.

For insulin I do a 1:2 dilution

For leptin I do 1:20 dilution.

Hope that helps.

Depending on what are you measuring you will find "activation" even in the absence of ·activator·. Then run controls without activators and , very important, treat cells as gently as possible.

Hello everybody,

I was thinking of conducting a study that investigates both pyscho-social well-being and biosphysiological markers that correlate with the well-being of students. There is some effort on the correlation between childhood obesity, life style and the prevalence of early adulthood diabetes. Additionally, other markers could be important, such as BMI, leptin, and even vitamin serum levels (such as B12 and D).  This is a rich area of investigation. But we can determine our priorities first and then decide on the variables which can be included in the study.

Adiponectin, Resistin, IL-8, VEGF, MCP-1, CRP, Insulin, IL-1β, MPO, MMP-9, IL-12P70, IL-4, IL-6, IL-13, TNF-alpha, IL-10, IFN-gamma,Leptin, Ghrelin, and IL-17A are used as salivary bio-markers to evaluate metabolic changes associated with obesity

Insulin resistant, maybe, but then you would see the inverse, no? High insulin with normal to high glucose. Sounds rather like a defect in insulin production or secretion. Or there is some problem with insulinotropic hormone production in the intestine. Interesting phenotype...

1.Leptin concentrations in relation to overall adiposity, fat distribution, and blood pressure in a rural Chinese population.

(PMID:11244467)

2.Paper: STUDY OF CORRELATION BETWEEN SERUM LEPTIN LEVELS AND BODY COMPOSITION IN HEALTHY OBESE AND NON-OBESE WOMEN

Author(s): OSTAD RAHIMI A.R.*, ZARGHAMI N.A., MORADI TALA, RAF RAF M.

3.Body Composition, Visceral Fat, Leptin, and Insulin Resistance in Asian Indian Men

Mary Ann Banerji, Nuzhat Faridi, Rajesh Atluri, Rochelle L. Chaiken, and Harold E. Lebovitz

- See more at: http://press.endocrine.org/doi/abs/10.1210/jcem.84.1.5371#sthash.CAgTeNMU.dpuf

Hope this might be useful...

Dear Juliana,

have a look at the attached files. These will give you a general introduction to RT-qPCR. This will explain a few things, such as how to validate your reference genes (ideally you would use more than one) and should also help you to understand the answer to your question.

There is no problem in running the two PCRs separately (they are two separate PCRs anyway!) and you are doing relative quantification. As long as you amplification efficiencies for both PCRs are close to 100%. You are using the reference genes to compensate for differences in RT-amplification, pipetting, RNA quantity and so on between your samples. Ideally the reference gene expression levels don't change between your treatment and control - however, you can't just assume that this is the case, therefore you need to validate your reference genes first.

Also, actin may not be your best option as it is fairly highly expressed in the cell and leptin probably quite a bit less. Ideally you would have reference genes that are fairly similarly expressed...

Hi,

You can go for Leptin ELISA kit from R&D systems.

You can query a database of sequences of 36 different mouse strains for your sequence of interest here:

Even if the mouse strains you are interested in are not in this database, you should be considerably reassured that there is not a likely difference between them, if there is no variation that has been detected in the 36 mouse strains that have been sequenced and compared.

@Mr. Asturiano: thank you very much..

@Mr. Kelany: it is interesting

Dear Mohammed, a PubMed search or a search on Google Scholar with the terms "glucocorticoids  AND (ghrelin OR leptin)" will provide you multitude of articles. Pleae find a short list with publications attached.

Here's a paper that might be useful:

Lu, X. Y. (2007). The leptin hypothesis of depression: a potential link between mood disorders and obesity? Curr Opin Pharmacol, 7(6), 648-652.

The full text is available here:

Dear Mojtaba,

Research on depression with inflammatory markers has grown considerably in the last 25 years. Leptin is only one of several important markers. I would also examine c-reactive protein, a correlate of crp, because  it is now used as for clinical diagnoses in many countries. Like leptin, crp is related to obesity, depression, plaques in the brain ( dementia) and certainly diabetes. It may be difficult to research leptin without considering crp or even insulin, which is related to anger and depression.

I currently have an undergraduate honors student who is completing his thesis. It shows that psychological distress (depression, anxiety) mediates increases in exercise in persons caring for a spouse with Alzheimer's disease, but not in matched older adults who are not caregivers.

Please be encouraged to pursue this research as it has great clinical meaning.

Here is an example with many good references.

Med Sci Sports Exerc. 2011 Jun;43(6):1002-9. doi: 10.1249/MSS.0b013e3182059eda.

Sixteen weeks of exercise reduces C-reactive protein levels in young women.

Arikawa AY1, Thomas W, Schmitz KH, Kurzer MS.

There is no easy way.  However, an increase in Leptin levels is indicative of Leptin resistance.  This is only correlational though.  The only way would be to look at downstream targets of activated leptin receptor in neurons expressing leptin receptor. 

Kulvinder,

A possible animal model for your POC efficacy study could be the diet induced obesity model using Nnt-mutant C57BL/6J (B6/J) (Jackson Laboratory number #664).  This animal model exhibits significant weight gain by 20 wks when fed a high fat (60% of calories).  The reference for the article is:

Diet induced obesity in two C57BL/6 substrains with intact or mutant nicotinamide nucleotide transhydrogenase (Nnt) gene.  Obesity 2010 18(10):1902-1905 the article comes from Edward Leiter's lab at Jackson.  Another rodent model might be the Zucker fatty rat (single gene mutation).  Hope this is of help to you.

Best,

Jorge L. Jacot, Ph.D.

Laura,

Here is a paper that shows that muscle mass contributes to whole body leptin production. 

You are likely to get less inter-animal variation if your rats were fasted.

The reason we use fasting is to eliminate some of the variability between different animals. When you leave rodents with unlimited access to food, not all of them eat at the same time or even the same amounts. If you have multiple animals in a single cage, you will often notice some differences in their weights. All of this plays into what you may see in serum levels of various measurements, especially parameters that are known to be effected by feeding status, such as the ones you are measuring.

If your experiment requires levels from animals that fed ad libitum, that's a different matter.

How did it work?

Hi,

I do not have any reference in human but you can refer to this article it can help.

Development of high fat diet-induced obesity and leptin resistance in C57Bl/6J mice. S Lin, T C Thomas, L H Storlien and X F Huang. May 2000, Volume 24, Number 5, Pages 639-646.

They established a scale to determine the leptin resistance rate you can maybe translated to human values.

Good luck.

With all due respect, there is no reasonable way that one could argue that the so called therapeutic effect of ECT is not actually the result of the brain trauma caused by inducing a grand mal seizure (of course in any other circumstance considered the sign for a severe medical disorder, epilepsy) and very damaging. I strongly recommend for the relevant overview of research that y'all review psychiatrist Peter Breggin's material on ECT research in the 2nd edition of his 2007 book Brain Disabling Treatments in Psychiatry.

Best.

Tomi

Does the leptin antibody block the leptin receptor or just bind with circulation leptin?