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# Glucose Metabolism - Science topic

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I noticed 13% -1Corbon, 27% -2 Carbons, 15%-3Carbons, 10%-4Carbons and 4%-5Carbons coming from Glucose to Glutamate in the TCA cycle noted by isotope tracer studies. Please explain the mechanism of how it happened.
There is no exact stoichiometry for any large cycle in metabolic networks. You can write out the system of equations for all the potential metabolites, then you need to add the recycle equations (including oxidative phosphorylation and PMF build up from NADH recycle). You will end up with more equations than unknowns, which implies an infinite number of solutions. To this system you need to add the sum of the free energies of all the reactions Delta Gr ≤ 0 to get the reactions to proceed, again, the inequality isn't sufficient to define a unique solution, only a boundary beyond which certain stoichiometries can't exist. This issue is called metabolic uncoupling. An example of this (for mixed acid fermentation), which explains the issues in biochemical stoichiometries, is provided in Schneider, LV, Biological Engineering: The unit operations and mathematical modeling of biology, Chp 2, (LVS Sciences, Houston, 2022). You can also look at the online lecture from this book (https://www.youtube.com/watch?v=6r9MVzwxdAs)
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I am interested in determining the amount of glucose that is taken up by a couple of cancer cell lines. I have tried a couple of commercial kits to no avail, so am attempting to use NBDG and measure it by fluorescence as has been done in the past. I have read that both 6-NBDG and 2-NBDG can be used for this purpose. My understanding of why 2-NBDG would be good is so that the the 6 position can then be phosphorylated (by hexokinase), not metabolized any further in glycolysis thus is retained in the cell (the theory behind 18F-FDG PET imaging), but I have also read that it does in fact get metabolized further - although I cannot find anything specific in the literature discussing this. On the other hand 6-NBDG is taken up by the cell but cannot be phosphorylated by hexokinase as it already modified at the 6-position. Would this mean that the 6-NBDG can then be transported back out of the cell and thus cannot be used to determine the amount of its accumulation inside the cell? Thank you for your input.
I have always been extremely wary of NBDG derivatives, see the recent excellent paper " Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters Kathryn E. Hamilton, Miranda F. Bouwer, Larry L. Louters, Brendan D. Looyenga Biochimie 190 (2021) 1-11." The large fluorescent 7- nitro-2,1,3-benzoxadiazol-4-yl-amino side chain is quite hydrophobic and thus enables to the glucose derivatives with this label to cross lipid membranes in parallel with any interactions with glucose transporters. So as well as the chemical acronym that NBDG usually describes, it could also be appropriately described as No BlooDy Good!
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Hi to all!
I am currently working on sequential glucose and oxygen delivery in tissue engineering field and I need to choose specific tissue and cells to work on. I wonder that which tissue need the highest amount of glucose during the first days of implantation. is there any valid reference reporting concentration of normal glucose consumption rate in different tissues?
As for as the oxygen demand is concerned the heart, followed by kidneys, brain and liver tops the list. The glucose demands is high for the brain. The demand is dependent upon the metabolic rate. The cancerous tissue also has higher demands for the glucose.
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I gave C57BL / 6 mice a solvent containing 17% DMSO by 0.1 mL i.p injection for 28 days. I saw that their weight and blood glucose levels decreased. Does anyone know the effect of DMSO on glucose metabolism?
There is no direct engagement of DMSO in the metabolism of glucose but DMSO with high doses promotes different body responses which are mostly toxic leading to an increase in the consumption of glucose.
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If kits would be used for determining the adiponectin levels, kindly provide details of how I can get the kits.
Okay Irfam
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Several studies showed that circulating osteocalcin level was associated with glucose/fat metabolism in humans [1,2]. Zhou, et al., showed that serum osteocalcin was inversely correlated with blood sugar and positively correlated with insulin secretion in the Chinese population [2]. Other researchers have also found a correlation between osteocalcin and glucose metabolism in humans [3,4], Previous clinical studies demonstrated that uncontrolled Diabetes could reduce serum OC level, while serum OC increased after blood glucose was well controlled [5] These data indicated that changes of glucose metabolism could influence OC levels [6]
[1] Kanazawa, Ippei, et al. “Serum osteocalcin level is associated with glucose metabolism and atherosclerosis parameters in type 2 diabetes mellitus.” The Journal of Clinical Endocrinology & MetabolismVol. 94, No. 1, 2009, pp. 45-49.
[2] Zhou, Mi, et al. “Serum osteocalcin concentrations in relation to glucose and lipid metabolism in Chinese indi-viduals.” European Journal of Endocrinology Vol. 161, No. 5, 2009, pp. 723-29.
[3] Fernández-Real, Jose Manuel, et al. “The relationship of serum osteocalcin concentration to insulin secretion, sensitivity, and disposal with hypocaloric diet and resistance training.” The Journal of Clinical Endocrinology & Metabolism Vol. 94, No. 1, 2009, pp. 237-45.
[4] Im, Jee-Aee, et al. “Relationship between osteocalcin and glucose metabolism in postmenopausal women.” Cli-nica Chimica Acta Vol. 396, No. 1, 2008, pp. 66-69.
[5] Motyl, Katherine J., Laura R. McCabe, and Ann V. Schwartz. “Bone and glucose metabolism: a two-way street.” Archives of Biochemistry and Biophysics Vol. 503, No. 1, 2010, pp. 2-10.
[6] Wang, Qingqing, et al. “The relationship between serum osteocalcin concentration and glucose metabolism in patients with type 2 diabetes mellitus.” International Journal of Endocrinology Vol.2013, 2013.
Bone is recently isrecognized as an endocrine organ. Accumulating evidence has shown that osteocalcin, which is specifically expressed in osteoblasts and secreted into the circulation, regulates glucose homeostasis by stimulating insulin expression in endocrine pancreas and adiponectin expression in adipocytes, resulting in improving glucose intolerance. On the other hand, insulin and adiponectin stimulate osteocalcin expression in osteoblasts, suggesting that positive feedforward loops exist among bone, pancreas, and adipose tissue. In addition, recent studies have shown that osteocalcin enhances insulin sensitivity and the differentiation in muscle, while secreted factors from muscle, myokines, regulate bone metabolism.In this regard, bone metabolism and glucose metabolism are associated with each other through the action of osteocalcin on bone, pancreas, brain, adipose tissue, and muscle.
Best
Zoheir
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There is a link between ion transport , action potential and glucose metabolism in the brain.
Sounds like somebody is asking us to do their homework!
Where does the energy for transmembrane transport come from? What is the function of the Na/K- (and Mg/Ca-) imbalance across the plasma membrane?
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Trying to look at glucose metabolism in HEPA cells, get the feeling that expression in this cell line is overall low. Eventually when converting to cDNA and looking at expression via qPCR all genes of interest were flatlining.
But still there is a paper where they looked at similar genes (albeit expression is shown to be low).
Does anyone have any tips? Could something simple as increasing the cell number (bigger dishes) increase my chances of successfully looking at these genes? Thanks in advance
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Hello,
Can anyone please give me some links to some good websites, videos papers etc on how metabolites affect CHO cells in bioreactor production for the following metabolites:
Glucose
Glutamine
Glutamate
Lactate
Ammonia
questions like how does each of these affect the cells and what can they tell us?
Any information anyone has would be greatly appreciated as all the papers I am finding online seem to be very specific. I'm trying to put a short 10 min presentation together. Thank you!
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Although it is well documented that glucose metabolism in the brain is reduced by alcohol consumption, especially in heavy drinkers, it is unknown whether alcohol affects glucose levels in the brain.
We want to know any one has reported the change of glucose levels in the brain after acute and chronic administration of alcohol in rats.
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I am planning an lab study on effect of hypoxia on glucose metabolism of cancer cell.
For hypoxia exposure, I am considering anaerobic chamber system.
My concern is that if I use this chamber, the cells have to be exposed to room air after pulling out of the anaerobic chamber and before being put back into the incubator (probably 10-15 min).
Would effect of hypoxiabe neutralized, if the cells (possibly cancer cell such as PC9) were exposed to room air for short while? I read a study result which showed that product such as HIF can be greatly influenced from such factor.
Is there any preventive measure I can refer to?
I hope the description is detailed enough to deliver necessary information. Any advice or comment is welcome.
Thank you.
Hi, Jeong
I don't know exactly what you want to analise after the hypoxia period, but if it includes HIF1a expression, you can also try to apply a drug that simulates the hypoxia effect by stabilizing HIF1a (inhibits its degradation). This also works if you are mostly interested in HIF1-mediated effects on your cells. The prolyl hydroxylase inhibitor dimethyloxaloylglycine (DMOG) is used for this purpose, and you can use it as a positive control to check if it alters the parameters you want to analise in your cells. Cobalt chloride is also used as inducer of hypoxia-like responses and you can play around with this substances depending on which parameters you are interested to check.
If you want to evaluate other proteins after the hypoxia period, HIF1a may not be present anymore, but some effects of the hypoxia (and HIF1a-mediated alterations) may be maintained. Some studies have demonstrated that 1h of hypoxia exposure is enough for astrocytes in cell culture to increase gene expression of HIF1a-modulated proteins. I checked this effect 24h after 1h-hypoxia exposure and the modulation of the parameters I wanted to see was still detectable by RT-PCRq. So, even if the cells are exposed to air room, the hypoxia effects may not be completely annulled, depending on what you are looking for. Also, you have to see if it's of interest to evaluate the cells after this 'reperfusion' paradigm. If not, prolonging your hypoxia time and analising it right away after taking them out of the chamber would be the best option.
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We are working on preparing a student lab. manual with the supplies in hand. To determine how much of the glucose is metabolized by bacteria in a broth, DNS method is recommended. The problem is we do not have 3,5 dinitrosalicylic acid in our lab, which has forced me to search alternative methods. Can you recommend any other spectrophotometric method we can use instead of DNS?
There are many methods for estimating reducing sugars..You can use following method-
Determination of reducing sugars by Nelson-Somogoyi method....Nelson (1944)
This is quite reliable method.
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I want to explore the effect of different stimuli on brian glucose metabolism in mice. Now I have series of PET/CT images from microPET/CT scan. I can see the differences from these images but I can't tell the specific brian regions. Due to the small volume of mouse brain, I cannot find a proper software for 3D reconstruction. Can anyone give me some help about analysing these data? Thanks so much.
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Rui X. Tian, Ph.D. (ruitian@yeah.net)
For decades, major treatments of cancer patients include surgery, radio-chemotherapy, anti-tumor targeted drugs and immunotherapy. Anti-tumor targeted drugs are primarily based on tumor DNA mutations, for example, anti-HER2 antibodies, EGFR inhibitors, etc. Actually kinase inhibitors account for a large proportion of anti-tumor targeted drugs. However, it is well known that cancer cells mutate rapidly, which makes drugs targeting a specific mutation not effective any more. This could potentially lead to a dilemma where cancer cells might outrun anticancer drug R&D.
Over ninety years ago, the Nobel Laureate O. Warburg described that cancer cells exhibit a different metabolism pattern. In contrast to normal cells ‘s oxidation-phosphorylation and TCA as the main energy source, cancer cells show increased glucose uptake, yet they primarily rely on glycolysis (lactate fermentation) even under sufficient oxygen supply. The Gonzalez et al. (2018) paper recently reported that mannose, a common monosaccharide, could inhibit tumor growth both in vitro and in vivo thereby enhancing chemotherapy. They found that mannose interferes tumor cell glucose metabolism not by competitively intake into cells against glucose but rather by accumulation of mannose-6-phosphate, which impairs glycolysis and other pathways of glucose metabolism. They also proved that lower levels of phosphomannose isomerase in cells are associated with mannose treatment sensitivity.
In accordance with the Gonzalez et al. (2018) paper, our interrogation of TCGA datasets, out of 499 head and neck cancer patients, higher expression of MPI (the phosphomannose isomerase encoding gene) is associated with worse prognosis. Patients of lower expression of MPI (370 patients) show a 5-year survival rate 50% while the number for patients with higher expression of MPI (129 patients) is 32% (p value <0.001). This result suggests that phosphomannose isomerase as many other key enzymes involved in glycolysis might be feasible for anti-tumor drug targeting. The philosophy underneath these potential new drug targets is to starve or constrain the growth of cancer cell rather to kill them. No matter how cancer cells mutate somatically, their way of metabolism follow the Warburg effect rule, that is where new drugs could ambush.
Reference:
Warburg O.The metabolism of carcinoma cells.J. Cancer Res. 1925; 9: 148-163
Gonzalez PS, O'Prey J, Cardaci S, Barthet VJA, Sakamaki JI, Beaumatin F, et al. Mannose impairs tumour growth and enhances chemotherapy. Nature. 2018 Nov;563(773 3):719-23. PubMed PMID: 30464341
In 1924, Otto Warburg discovered that tumor cells tend to produce large amounts of lactate from glucose, regardless of the available oxygen level [15, 16]. This situation is similar to anaerobic glycolysis, implying that oxidative phosphorylation (OXPHOS) is replaced by glycolysis in normal differentiated cells under hypoxia [23, 24]. However, cancer cells appear to engage in glycolytic metabolism before they are exposed to hypoxic conditions [15, 16]. OXPHOS in mitochondria generates as many as 36 mol ATP from 1 mol glucose, whereas the conversion of glucose to pyruvate or lactate produces only 2 or 4 mol ATP, respectively [25, 26]. It remains unclear why cancer cells largely depend on this “inefficient” metabolic pathway, even when enough oxygen is available [27, 28]. In striking contrast to normal cells, cancer cells preferentially uptake and convert glucose into lactate even in the presence of sufficient oxygen [29]. This seemingly “inefficient” metabolic characteristic relies largely on aberrant upregulation of GLUT1, a glucose transporters abundantly expressed in cancer cells [30, 31], although one contradictory study reported that GLUT1 is not necessarily involved in the Warburg effect depending on the degree of tumor invasiveness [32]. Inefficient ATP synthesis becomes an obstacle for cancer cells only when their energy resources are scarce. However, this is not the case in proliferating cancer cells with aberrant angiogenesis [29]. Tumor cells finely regulate ATP synthesis by regulating substrate uptake, as well as enzymes related to glycolysis, which enables them adapt to the nutrient microenvironment [33]. Moreover, the regulation of adenosine monophosphate-activated protein kinase (AMPK) signal transduction, a sensor of energy status, is intimately connected to the Warburg effect, one form of metabolic reprogramming of cancer cells [34, 35]. Indeed, genetic ablation of AMPK activates mammalian target of rapamycin (mTOR) signal with ectopic expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha), resulting in rapid cellular proliferation accompanied by activation of aerobic glycolysis [35]. This strongly suggests the importance of cancer metabolic reprogramming in maintaining the interaction between the oxygen-sensing transcription factor and the nutrient-sensing signal pathway.
Cited from
Metabolic reprogramming: the emerging concept and associated therapeutic strategies
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Is the conversion factor 6.945 or 6?
Actually, it is 6. This seems to be a very common area of confusion for a long time, but the literature suggests 6 is the correct conversion.
The American Diabetes Association gives the conversion as 6 in their tables of unit conversions: http://care.diabetesjournals.org/content/21/12/2206
No idea where the 6.945 came from, I'm currently trying to hunt down the origins of that. Any help would be appreciated!
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Dear Professor and colleagues:
inflammation and cell proliferation induced via activation of nFKappaB leading to an increase in the transcription of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6...)
in case of abnormal metabolism, cancer cells need inflammation to growth and to form a new blood vessels "neoangiogenesis" which will supply cancer cells by nutrients and oxygen... thus, to the spread of tumors to other organs which we call "Metastasis".
In the other hand, Sugar supports this mechanism, but how? I mean what are the specific signaling pathways that lead to increasing Glucose metabolism during aerobic glycosis (Warburg effect)!!!??
Could anybody help me to know what are the most signaling pathways involved in the metabolic pathways...
Thank you very much for you valuable answers,
Sincerely,
Fouzia
GSK3beta is negatively downregulated signalling moleculue. That means, when GSK3beta is phosphorylated at serine 9 in presence of signals like Akt/PI3K/PKA/Wnt , they participate in metabolic syndromes.
As you have mention' GSK3beta induces inflammation and cell proliferation via activation of nFKappaB leading to an increase in the transcription of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) '
''it means all these sequences are happening when GSK-3beta is unhpshorylatd/activated, so we need to phosphorylate/deactivate GSK-3beta in order to avchive anti-inflamatory effects which is prequisite in metabolic syndrome. Additionally GSK-3beta has shown to mitigate insulin resistance, which again confirm that inactivation/phophorylation of GSK-3beta is prerquisite for acting against metabolic syndrome.
GSK-3beta act by phosphorylation/deactivation of GSK-3beta at ser9 domain.
in brief, Akt/PI3K/PKA/Wnt and as mentioned by John Patrick Alao, mTOR pathways should be considered
You may refer to : Song WJ, Song EA, Jung MS, Choi SH, Baik HH, Jin BK, Kim JH, Chung SH. Phosphorylation and Inactivation of GSK3β by Dual-Specificity Tyrosine (Y)-Phosphorylation-Regulated Kinase 1A (Dyrk1A). Journal of Biological Chemistry. 2014 Dec 4:jbc-M114.
regards,
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I’ve followed recent published papers to measure glucose uptake in HepG2 cell. The problems I found that (1) All of the fluorescent values obtained are similar. I think my protocol is wrong that was (cell culture with 10% FBS MEM + after 24 hr media suction and treated insulin 100nM with SF-MEM to produce insulin resistance for 24 hr + again suction media and treated with sample in different concentration for 24 hr + Media suction and add insulin for 30min + media suction treated with 50μM 2-NBDG for 30 min with SF MEM+ wash media 2 times + fluroscence 485nM excitation and 528 emission). Does anyone have an exact protocol I could follow where these problems don't occur?
Hi Bhakta,
Your papers were very helpful, however, i still have a question related to this assay: in my experiments, i don't want to induce insulin resistance - so I removed from my protocol the 24h incubation with insulin that you have done.
In my experiments I just want to see if my insulin can induce glucose uptake in HepG2 cells. So, should I starve my cells overnight in DMEM FBS-free, and them incubate cells with insulin and 6-NBDG at the same time for 20-30min and then stop reaction, lyse cells and measure fluorescence?
Cheers,
Viviane
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Is it possible for insulin sensitivity to change within 5 hours of consuming an antioxidant nutritional intervention?
I completly agree that insulin sensitivity is very dinamically altered by many physiologically or pathologic conditions. In dogs, secondary diabetes as result of higuer progesterone and GH levels is very commom during diestrus in intact females. When we remove the source of those antagonic hormonal stimuli, many bitches achiev diabetes remission, sometimes few days after surgery. This paper may help...
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Dear, colleagues!
Is it possible to find labs (better in Europe), where scientists explore insulin pathways and actions on a cell protein or glucose metabolism and how they interact with drugs, hormones or other active substances?
Thank you for you attention!
I think that Angel Nadal that is one PI that currently is working in the University of Elche can help you. I made some experiments at his lab and they are experts in ex vivo and in vitro experiments.
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C2C12, 3T3-L1, L-6 and HepG2: among these, which cell line will be ideal for in vitro glucose uptake activity?
@Sunday Oyedemi Just a correction, but C2C12 are not intestinal cells. They are mouse skeletal muscle cells.
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Hello
I am looking for a rat somatostatin (SST) 96 well plate ELISA with a required sample (plasma) volume of 10-20 µl, preferably from a US/Canada based supplier. All SST ELISA kits that I have been working with so far require a sample volume of 50µl in duplicates.
I know that in the case of small volume samples, original samples can be diluted to solve the problem, but I'm trying to avoid dilutions as much as possible, so instead I'm hoping to find an alternative ELISA with the minimum undiluted sample volume required.
ELISA kits are cheaper, less time consuming and much safer compared to RIA kits, so they are usually preferred over RIA. Also, as far as I know, RIA kits are not common for rat somatostatin at least.
As for EIA, I believe ELISA is basically a form of competitive EIA.
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I have drink with fructose in it.
I expect the fructose to spike insulin. After secretion of insulin, should I expect to see increased translocation of GLUT5?
If GLUT2 is insulin independent, presumably this would not have any effect on insulin secretion.
GlucT2 is located in cell membranes but GlucT 4 and 5 are intracellular, then expressed and translocated by insulin, since are insulin dependent.
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Hi,
I have a decrease of 1/3 to 1/2 in the gene expression of gluconeogenesis enzymes (Glucose-6-phosphatase, Fructose-1,6-bisphosphatase, Phosphoenolpyruvate carboxykinase and Pyruvate carboxylase) in mice liver between my test condition and my control condition and I would like to know if it can lead to a reduction of the flux of the pathway or not? Also is one of these enzymes more critical than the others in the pathway?
Thank you.
In addition to what my colleagues have stated, One may actually claimed inhibition of gluconeogenesis however protein quantitation would brings out the beauty of the work and a better conclusion. Thanks
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heart metabolises lactate has LDH B , but liver  which also metabolises lactate has LDH A
Production of glucose is a very important function of the liver but is not the sole function regarding pyruvate use and ATP production. The hepatocyte needs a lot of energy obtained in the mitochondria and thus must convert a sustantial part of pyruvate to acetyl-CoA. It is not convenient to drain much pyruvate to glucosa unless urgently needed. Regulation of pyruvate metabolism is highly complex.
The answer for the question you pose is unkown, I guess. But evolution seems to be clever. You could have a look at the combination of transcription factors that act on the genes coding for the LDH subunits.
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It is know that glucose can generate ATP for energy via biological metabolism. Since most chemical reactions are accompanied by heat release, I am curious if it is possible to transform glucose into heat via some enzyme or other chemical reactions at a high efficiency? Technically, I mean generate heat, but not burn off glucose by heating.
Yeast fermentation of glucose will generate heat energy without burning off the glucose by direct heat.
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Which is more accurate among all commercially available personal glucometers to measure glucose concentration in water-glucose samples? Is there any rating available for personal glucometers based on the performance?
Hi Satish ¡ I agree with Tausif obout the use of blood glucometers  for measurement of glucose in wáter. You can be sure with the measurement of glucose in kown concentrations in wáter on   several  solutions prepared by yourself
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I wish to evaluate insulin receptor density and affinity following different glucose and insulin concentrations. As several thyroid cancer cell lines are available in literature (human model), how would you base your screening and finally choose? I am not familiar with cell cultures, this is why I would start with cancer cells.
Why not check the expression level of TSH receptor?
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The insulin secretion in the body of a Type 1 Diabetic is impaired/absent. Is there any insulin secretion in the body for a typical Type 1 Diabetic Patient? If yes then what is the level (typical value)?
The context of the question is that when we simulate the compartmental model of Type 1 Diabetic patient, then is it necessary to include some constant basal value of insulin? But for control problem of blood glucose regulation of Type 1 Diabetes we sometimes assume that the body produce zero insulin.
Summarizing the query, it is that should we include some basal insulin concentration in blood plasma or consider it as zero.
The answer to your question, as it is put, depends on the stage of T1DM along its temporal progression. If you review the literature, you will see that early on in the disease, there are some islets but eventually they are all destroyed.
A C-petide measurement on a specific patient will give you a much better idea as to how much insulin is coming from endogenous remaining islets at a given point.
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What is the chance of UDP-alpha-D-glucose and UDP-beta-D-glucose formation in One-Pot in vitro reaction catalyze by series of enzymes from beta-D-glucose as primary precursor.
Is there any chance of changing anomeric forms of glucose (change in beta-D-glucose when alpha-D-glucose was used as precursor sugar and vise versa)?
Is there any effect of acidic or basic solutions over change in anomeric forms of glucose during reaction catalyses?
An enzyme that starts with a beta anomeric form of a substrate and ends up making a beta-anomeric form is called a retaining-type of enzyme, since the anomeric form of the substrate is retained in the product. If it begins with an alpha-anomeric form of the substrate and ends up making a a beta-anomeric form of the product, it is called an inverting-type of enzyme. Whether an enzyme is retaining or inverting depends on its mechanism, which depends on its sequence and structure. Both types exist in nature, although I'm not sure if that applies to your particular enzyme. To respond to your question about making UDP-beta-D-Glucoside from free beta-D-glucose, you need some energy source to produce the high-energy UDP-Glc bond, regardless of which anomeric form of glucose is present. That typically occurs by coupling of the synthetic reaction to hydrolysis of some other high-energy bond, like a sugar phosphate, etc.
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I want to grow E. coli in a fed-Batch culture. The idea is to add Glucose when the bacteria stop using oxigen (pO2 > 50%).
How can I make sure that a high pO2-level is really due to a lack of Glucose and not caused by other reasons (stationary Phase of the bacteria, too high Input of air, reduced Glucose metabolism...)
Dear Marius,
my question concerns a third Dimension:
I want to Change the Speed of the feed pump so that Glucose will only be added when the Glucose in the medium is used up. Lack of Glucose in the medium results in a steep increase of the pO2-level. I can use this Signal to regulate the feeding rate. However it may happen, that the pO2-level rises for other reasons other than lack of Glucose. I am looking for an algorithm to check that..
Rolf
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When investigating the underlying mechanisms of glucose uptake promoting effects, the GLUT4 levels of plasma membrane (PM) and cell lysate (CL) are determined in order to assess the GLUT4 translocation into the plasma membrane. Here, the insulin receptor beta is used as an internal standard to determine the GLUT4 levels in the PM and CL, and I want to know the reason behind this logic.
I wouldn't use Insulin receptor as an internal standard. As Barnabe mentioned the Insulin Receptor is internalised after insulin binds to it, therefore it is not a good loading control. I would use actin or GAPDH. You can use insulin receptor as a PM marker to make sure you have purified the PMs.
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I am trying to do GSIS using MIN6 cells and I am measuring secretion via ELISA; surprisingly I am obtaining higher insulin secretion when I am stimulating the cells with low glucose concentration (3.3mM) than with high glucose concentration (16.7mM).
In an ideal situation, one would perifuse insulin producing cells but that makes for a somewhat complicated experimental setup. In static incubations, one must always go back to low or normal glucose at the end to demonstrate that there is no (or insignificant) cell death or detachment (always gently spin the collected medium to remove detached cells), giving misleading numbers.
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I did a glucose stimulated insulin secretion assay for MIN6 cells. I measured the released insulin in the Kreb's Ringer buffer containing certain concentrations of glucose. In the end I collected the cell lysates of the treated cells using TNE buffer with 1%SDS. I aim now at measuring the insulin content in these collected lysates by ELISA.  My concern is about the TNE buffer with 0.1% SDS, I had a look over several articles I saw researchers extract proteins from cells using acid alcohol mixture or triton-X buffer. I wonder if I can use my cell lysates extracted in TNE buffer-0.1%SDS for determination of insulin content by ELISA.
The quick answer is no, it wouldn't work; do the acid acetone extraction like everyone else. In fact, you need not lyse cells in SDS. Simply freeze the cells and add pre-chilled (-20ºC) acid-acetone to extract and recover insulin.
It is unlikely that you will be able to get good binding of insulin to the primary antibody, and therefore accurate measurements of insulin by ELISA in the presence of 0.1% SDS.
Some antibodies can be more forgiving and at relatively low concentration (usually <0.05%) of detergents may bind their antigen but it is uncommon in the case of SDS. If on the other hand, you do happen to have such an antibody, make sure that your standards have precisely the same composition (including the detergent) as your unknown samples.
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why glucose increases, but insulin doesn't change?
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...
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The surrogate marker HOMA - IR which we apply to human beings cannot be applied in animal experiments, because the baseline glucose and insulin values differ for each species. Given this, how HOMA - IR can be calculated in swiss albino mice?
Dear,
In Swiss Albino mice, insulin resistance is estimated using the homeostasis model assessment for insulin resistance (HOMA-IR) index using the formula described previously by Mathew et al., HOMA-IR index = [fasting glucose (mmol/L) × fasting insulin (mU/ml)].
See this paper:
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As platelets have active PI3K/Akt pathway, I'm just wondering whether PI3K/Akt pathway would play a role in platelet glucose metabolism
I got the mTOR signal with human platelet protein by western blot with antibody from Cell Signaling Technology‎.
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I am following the method as described by Hughey et al (www.jove.com/details.php?id=2432) for conducting the hyperinsulinemic-euglycemic clamp in Wistar rats (400g) but the overnight fasted blood (arterial) glucose level varies considerably between rats, from 6-10 mM. Once I commence insulin infusion (2 uL/min, 4mU/kg/min) is it most appropriate to clamp the glucose level at the rat's baseline glucose level or at a fixed amount e.g. 5-5.5 mM?
n.b. the only major modification that we have made to the Hughey method is that we are conducting this procedure under pentobarbital anaesthesia (60 mg/kg), which according to Saha et al (2005; Exp Biol Med 230, 777-784) does not affect glycemia.
Sure, I have used both sevoflurane and isoflurane, with propofol for induction.
Also, my work is in dogs, which makes it easier to regulate food intake.  The blood glucoses are very consistent in conscious animals under fasting conditions, and we almost never detect hyperglycemia.  We do typically take glucose levels prior to anesthesia, and it does seem to be an anesthesia specific effect as they are not hyperglycemic prior to induction of anesthesia.
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Alloxan and streptozocin both poison beta cells producing elevated blood sugar levels supposedly causing microvascular disease, but both toxins may be capable of causing injury to the endothelial cells?   How to study this to pull the two variables apart?
The response to your question: "what caused glucose to become a vascular toxin? " is reduced membrane flexibility.
Phospholipid bilayers form rapidly and spontaneously when phospholipids are added to water. The two acyl chains yield a roughly cylindrical molecule that can easily pack to parallel arrays to form extended sheets of membranes composed of a mosaic of proteins and phospholipids in a fluid phospholipid matrix. (Poly) unsaturated acyl chains of phospholipid membranes have an intrinsic propensity toward cell membrane flexibility. Saturated fatty acids possess essentially linear acyl chains, with no double bonds. Conversily, double bonds in unsaturated fatty acids are nearly in the cis configuration, which produces a bend in the fatty acyl chain. This bend makes it more difficult for phospholipids with unsaturated acyl chains to pack close together, thus promoting bilayer flexibility. Various studies of fully hydrated, fluid phase, model phosphatidylcholine bilayers have demonstrated that introducing one or more carbon-carbon cis double bonds into the saturated acyl chains will produce an 8.5% increased interchain distance which results in a 33% decreased attaction energy per pair of fatty acyl carbon atoms and ,in turn, in a greater membrane flexibility. A number of papers have reported examples of reduced membrane flexibility. A review of Cho et al. indicated that patients with type 2 diabetes exhibited reduced erythrocyte deformability compared to healthy controls [1].
The erythrocyte membrane is compositionally very similar to the vascular endothelium. This has crucial implications because, in capillaries, the size of red blood cells is of the same order of magnitude as the capillary lumen (about 8 micrometer); thus, deformability is an important determinant of blood flow. Increased stiffness of both the microvascular endothelium and the erythrocyte membrane decreases the microcirculatory flow,which leads to reduced oxygen supply, and consequently, to chronic tissue hypoxia, reduced adenosine triphosphate production, and ultimately, increased endothelial dysfunction.
A characteristic of type 2 diabetes and its prediabetic phase, is an increased concentration of free fatty acids, which causes a shift from unsaturated to saturated fatty acyl chains in membrane phospholipids. Consequently, there is harmful increased membrane stiffness with a concomitant reduction in all Class I glucose transporters, which results in a decrease in glucose into cells. In turn, this would further stimulate hepatic li[poysis to aquire sufficient ATP via beta-oxidation of free fatty acids.
In conclusion, not glucose, but elevated free fatty acids cause endothelial dysfunction and vascular lesions in type 2 diabetes due to a shift from unsaturated to saturated fatty acyl chains in membrane phospholipids, including the cell membranes of islet cells.
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I would like to investigate glucose metabolism in cultured cancer cell lines with the use of D-[3-3H]glucose; the rate of glucose oxidation by means of measuring the trititated water derived from glucose and the extent of incorporation of glucose into lactate in extracellular medium.
For the part of glucose oxidation, we try to dry the medium and thus the trititated water would be evaporated and  the remaining cpm should be from glucose. However, we find that there is a significance loss of the cpm reading even we dry the freshly added tritiated glucose in the medium. Does anyone experience these kind of problems or if there are any alternative methods to do it?
Also, does anyone know are if there any simple methods to measure the incorporation of glucose into lactate apart from making use of MS or HPLC?
Thanks
The easiest way to measure the in vivo rate of formation of Lactate from glucose would be to directly measure the specific metabolites: glucose and lactate.  Use stable isotope isotopomer of glucose / lactose and use  a 300MHz+ NMR to look at the concentrations.  The MS could also be used but the NMR is ideal because metabolites can easily be isolated by methanol extraction or if you have a method of perfusing the cells, the reaction in the cells could actually be observed in real time.  Hope this helps
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Can someone suggest a genetically modified spontaneous mammary tumor /chemical induced carcinogenesis model where genetic modification should result in significant alteration in glucose metabolism in mammary epithelium!
Thank you very much.
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I'm trying to select one assay to measure glucose uptake/ utilization in beta cells/islets. Do you know which one is more accurate to use, a conventional radioactive using tritium label or colorimetric kit by Abcam? I want to know if I can achieve the same result without using radioactive material. Thanks
I worked with Biovision Glucose assay kit (Catalog #k606-100). We used the Fluorometric assay and could detect glucose in saliva. Please see the paper: Hartman M-L, Goodson JM, Barake R, Alsmadi, O, Al-Mutawa, Ariga, J Soparkar Behbehani J, Behbehani K, Welty, F. 2014 Salivary glucose concentration exhibits threshold kinetics in normal-weight, overweight, and obese children. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2014:8 9-15. I did not compare this glucose test to the radioactive one therefore I don’t have an information on that.
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I just got the primary cell line of HK-2 from supplier, but I don't have the Keratinocyte Serum Free Medium which is recommended by ATCC. I managed to find some papers in which they use DMEM/F12 to culture the HK-2 cells. I really want to know how to prepare this DMEM/F12 medium and any other things that I need to be aware of when I deal with this cell line. Thank you so much.
You can use low glucose DMEM/F12.
The routine media will be DMEM/F12 (high glucose) - 500ML DMEM/F12 commercially purchase from ATCC + 10mL penicilin streptomycin +50mL Fetal calf serum (FCS) + 1mL endothelial growth factor (EGF)
For treatmemt media - (low glucose) is just halfing your DMEM and F12., for optimization.
you prepare (DMEM component) = 500ML DMEM + 10mL PEN STREP + 50mL FCS + 10EGF
and
F12 component = 500ML F12 + 10mL PEN STREP + 50mL FCS + 10EGF
and then mix in the ration of 1:1 (say 10ML of DMEM + 10ML of F12 in a fulcon tube)
For Serum free media - which should work like keratinocyte serum free medium, prepare DMEM component and F12 component but DONT add FCS. then mix in the ratio 1:1 still.
NB: EGF must always be on ice.
Hope this help
If you can order directly from ATCC Keratinocyte serum free medium (K-SFM), it saves you time for preparation of DMEM/F12 serum free yourself.
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Our approved protocol states the tail clipping method with scab removal to monitor glucose levels in our rats. However, the University Vet walked in on a glucose test being done and wasn't happy with what is happening. This is standard from everything we've been able to find but she has officially made us cease research because of this method. I'm trying to compile as many papers as I can that supports us in this method. What do you have?
Do you maybe even have a better method for monitoring?
If you have to snip the tail end every so often and/or peel the scab, you are almost certainly going to end up with an infected tail at some point. if you think that snipping tail and peeling the scab daily for rats over an extended period of time is less stressful than a clean tiny needle puncture at the end of the tail, I can imagine why you are making your vet nervous.
We have gone well beyond the period you mentioned - without any infections or obvious signs of stress to animals (or the lab personnel). As I mentioned earlier, rats barely move and no strainer is required for the procedure. The entire process takes no more than a minute.
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I want to know activity of glycerate kinase from my microbial culture. I am not able to find out suitable method to do it properly. Please suggest me better protocol for analysis of this enzyme.
Hi there,
Glycerate kinase activity can be easily coupled to pyruvate kinase and lactate deshydrogenase. GK activity will result in the stoechiometric consumption of NADH measured at 340nm. For more info this paper should help :
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I am trying to search for specific cancer cell lines that have different levels (high and low specifically GLUT1&3) of GLUT cell surface membrane expression. If anyone has any knowledge of such database, I would appreciate it.
Use the Cancer Cell Encyclopedia: http://www.broadinstitute.org/ccle
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Currently we culture our MDA-MB-231 cancer cells in DMEM (containing 25mM glucose) with 10% FBS + 1% PenStrep. I am wanting to assess the effects of different glucose concentrations (25 mM and 5 mM glucose) on metabolic pathways (glycolysis and b-oxidation) in these cells treated with ketone bodies. I have seen that one way of assessing low glucose concentrations in these cells would be to culture them in 25 mM glucose, glucose starve the cells and supplement with pure glucose at 5 mM before each experiment. I am worried that this would induce metabolic alterations consistent with acute hypoglycaemia and does not accurately depict long term normoglycaemic conditions! Can anyone perhaps help me with information on how I can culture these cells in 5 mM glucose without inducing metabolic alterations that will affect my experiments?
Agreed with Tausif Alam
Prefer begin in two separate batches of cells.
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I wish to know that whether a person can have not very high fasting glucose but very high HbA1c at the same point of time.
As I already mentioned, and you already seem to know, a single fasting blood glucose level represents the situation of that specific moment on that day whereas HbA1c is the representation of the average blood glucose for a few months.
In the case of your example of FBG 310 and A1c ~7, you know that if even the average blood glucose was ~300mg/dl,much less the fasting, which should be lower than average, the A1c would be ~12. The only way these values are possible is through some other issues of which you were not aware, for example an infection (or something drastically changing about the status of patient's disease, such as loss of islet mass/function). Alternatively, the patient with abnormal GT could have not told you the whole story - after an overnight fast, felt hungry and on the way to your clinic couldn't resist sweets freshly being made!
Your first example is not too surprising, perhaps the person is in his or early phase of better management of diabetes and the lowered values are not yet reflected at the level of A1c. Alternatively, longer than normal fasting time.
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Is there any evidence to support that sulphonylureas burns out the pancreatic beta cells?
The kind of sweeping statements made by Marcia and others regarding "burnout" seem to be based on some sort of vague belief rather than any supportive evidence or science. I must agree with Tausif that we need to define our terms carefully.
I still maintain that sulphonylureas do not accelerate beta cell apoptosis and no-one in this discussion thread has provided any hard evidence to the contrary.
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I have injected 90 mg/Kg body weight of Alloxen as an IP dose to Rats for the induction of type 2 diabeties but all the rats died overnight. Could anybody suggest something for trouble shooting this issue?
I suggest you use an established model of type two diabetes. Chemical destruction of beta cells does not resemble type two diabetes. Please do not waste time,  money and animal lives on a poor experimental design.
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I would like to use a colorimetric assay to measure glucokinase activity from hepatic extract. Does anyone have experience using any commercial kit?
The most straightforward way is to use a coupled assay, where glucokinase converts glucose (at greater than minimally saturating concentration of glucose, 25mM or higher) to glucose-6-phosphate in the presence of ATP, which is then used as the substrate by the next enzyme glucose-6-phosphate dehydrogenase already present in the mixture along with other necessary components (NADP). NADP is quantitatively reduced to NADPH, kinetics of this reduction can be followed on a spectrophotometer and correlated to the activity of glucokinase, which is kept as the limiting variable. In the absence of a spectrophotometer, its possible to quantify the reduction reaction by a colorimetric set of reagents. I believe that many vendors sell these reagents in a kit form - for example Abcam. Be aware that to get accurate quantitation, like any other enzyme, you need the initial rate of reaction, corrected for proper controls.
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What does circulating PPAR gama mean? Is there detectable amount of this transcriptional factor in plasma? If yes what could be possible explanation?
There is a report indicated the presence of PPARg circulating in exosomes.
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We injected DOPA into the body of patient. We observed that the blood glucose reduced and urine glucose increased significantly.
Interesting finding.  Could be the dopaminergic system is negatively regulating SGLT2 to produce similar findings as with the new class of "flozin" anti-diabetic drugs.  Is the effect maintained over time?
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I made an acute knockout gene of B6, which ought to be somewhat related to glucose regulation. But acute KO seems lethal in 8 days.
So I witnessed that it became weaker and weaker, but the glucose levels seem to remain unaffected.
I assume that poor health might diminish its foraging behaviors, how can I prove that?
Your question is too vague to be answered easily. Perhaps you should begin with a description of the gene you knocked out and tell us about its function, if known, or postulated function. Only then can one design reasonable countermeasures or interventions that will have the potential to stabilize the weight and improve poor health of animals.
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I want to test the effect of small molecules on glucose uptake by differentiated L1 adipocytes and differentiated C1C12. I used the glucose assay kit (HK) from Sigma on the recommendation of a collaborator, and also tested the Wako Autokit. I don't see glucose concentration decreases in the media after treatment with insulin. I have serum-starved and glucose starved the cells up to 1h, pre-treated with 100nM insulin for up to 30min, and I am still unable to see a change in media glucose concentration. Anyone has a suggestion or recommendation?
Hi Celine!
Do you have facilities for isotope work in your lab? For adipocytes I always measure the uptake of 3H-labelled 2-deoxyglucose (2-DOG), the assay is pretty straightforward, you serum-starve them a bit, then let them adapt to low glucose Krebs-Ringer-Buffer (1g/L, i tried glucose free buffer, but this lead to cells that were so starved that all of them exhibited immense glucose uptake in the assay, independant of insulin), then I add the traced deoxyglucose plus 20nM insulin (i tested lower and higher concentrations, usually 20nM is more than sufficient) and after 15-20min you can usually see 2 to 3-fold stimulation of glucose uptake by insulin (at this point the reaction is stopped by cold buffer containing excess glucose (non-radioactive), washed, cells are lysed and scintillation counting is performed)
good luck!
Karin
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Does anyone know what is the "ideal' insulin concentration for insulin-stimulated glucose uptake in mouse soleus muscle explant?
For submax in soleus I use 100 or 200 uU/ml and for maximal stimulations 10000uU/ml.
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It is an epimerization reaction of glucose, I need a reaction mechanism.
Paper attached
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We learn in medical school that in normal conditions glucose doe not reach the urine. If we detect glucose in the urine how do we explain this?
I forgoot high fat diet also induce the typical changes which you would expect for the plasma, you see that also in urine.
so I hope that helps you a bit in your research. unfortunately those data are not puplished yet, but for deeper questions ask Prof. Daniel even here on this plattform!
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Many studies show during type-2 diabetes non insulin mediated glucose uptake will upregulate and represent a major part in the regulation of glucose homeostasis at normal condition also. Which are the main pathways other than the ampk pathway involved in non insulin mediated glucose uptake?
There is eveidence for two types of GLUT-4 transporters, a GLUT4IR - insulin regulatable and a GLUT-4EX or ER - exercise regulatable. The GLUT-4IR is located in adipocytes and myocytes, while the GLUT-4ER are located only in myocytes and are activated in response to exercise and glycogen depletion.
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Similarly study of glucose/sucrose transport in yeast will also be very helpful.