Glucose Metabolism - Science topic

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)

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!

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.

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.

Okay Irfam

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

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?

Hello

You will find answer in these links:

https://bitesizebio.com/2345/troubleshooting-rna-isolation/

http://www.geneaid.com/faq/how-do-i-increase-yields-total-rna

https://www.researchgate.net/post/How_to_increase_concentration_of_RNA_and_why_do_the_ration_of_A260_A230_is_low_and_how_to_overcome_them

Good Luck

Follow

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.

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.

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.

@ Yuan Li: Please do check the below link

Moreover, I am sending you a ebook which may help you

Thanks

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

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!

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,

Hi Bhakta,

Thank you very much for your reply.

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?

Also, what incubation solution your cells will be when adding 6-NBDG?

Cheers,

Viviane

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...

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.

@Sunday Oyedemi Just a correction, but C2C12 are not intestinal cells. They are mouse skeletal muscle cells.

Thanks for your input Molly!

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.

GlucT2 is located in cell membranes but GlucT 4 and 5 are intracellular, then expressed and translocated by insulin, since are insulin dependent. 

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

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.

Yeast fermentation of glucose will generate heat energy without burning off the glucose by direct heat.

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

Why not check the expression level of TSH receptor?

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. 

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.

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

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.  

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. 

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.

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...

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:

I got the mTOR signal with human platelet protein by western blot with antibody from Cell Signaling Technology‎.

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.  

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.   

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

Thank you very much.

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.

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.

Adam,

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. 

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 :

Use the Cancer Cell Encyclopedia: http://www.broadinstitute.org/ccle

Agreed with Tausif Alam

Prefer begin in two separate batches of cells.

see the article i hope help you .

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. 

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. 

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.

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.

There is a report indicated the presence of PPARg circulating in exosomes.

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?

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.

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

For submax in soleus I use 100 or 200 uU/ml and for maximal stimulations 10000uU/ml.

Paper attached

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!

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.

Yes, there are many different methods to determine the uptake of glucose by organism:

One simple method would be to determine the initial glucose levels either by biochemical or enzymatic methods (GOD-POD) and then determine the glucose level thereafter at different interval.

The other method would be to use radio-labelled glucose and then determine the uptake by determining the radioactivity in control and test samples.

If I may, it is a cheap compound these days: SA has it for 320$/g,

There is few methods available, by experience I would recommand, this one:

JOC2006, V71 p4565-77, using TMSN3/SnCl4/DCM

works really well. Done it on up to 50g scale. One step from the per O acetylated sugar (beta)...