Difference between revisions of "Talk:Glucose"

Latest revision as of 13:07, 8 March 2021

Former Instructions

These instructions were given until 2020-03-08. They were updated because the volume of the solute was not taken into account. Therefore the concentration oft the stock solution was lower than intended.

Preparation of 2 M stock solution (dissolved in H₂O):

weigh 360.32 mg of Glucose
dissolve 1 mL H₂O in 1.5 mL Eppendorf

Preparation of 2.2 M stock solution (dissolved in H₂O):

weigh 79.2 mg of Glucose
dissolve 0.2 mL H₂O in 0.5 mL Eppendorf

MiPNet discussion forum: glucose oxidation in skeletal muscle (2013-10-29)

Dominik Pesta

Has anyone ever looked at glucose oxidation in intact skeletal muscle fibres (of mouse muscle)? I have tried with a Krebs Henseleit Buffer (no substrates) and Malate and/or Pyruvate followed by glucose (basal) and insulin titration (insulin stimulated) but do not get any stimulation of respiration. Although malate will not permeate the cell, I would at least expect some stimulation with glucose. Any thoughts on that?

Rob Wuest

My initial thought on Dominik's question was that the mitos are quite likely not stimulated well enough with insulin to respire any of the glucose. I suspect that the insulin titration is activating the "glucose-entering" pathway, but that pathway quite likely does not consume enough ATP in sk muscle to quickly activate any of the mitochondria. There is enough PCr etc to buffer all this. The easiest way to increase VO2 in these fibres is to stimulate them to contract.

Renata Goncalves

Why would you expect an increase in the basal respiration of the intact fiber when you add glucose or glucose plus pyruvate? I would not expect an increase in the basal respiration just because there are more substrates available to be consumed. In muscle cells the control of the substrate utilization is not based on the substrate availability but instead in the activation of the dehydrogenases by Ca++ for example. To observe an increase in the respiration you should increase the ATP demand or increase the activation of the dehydrogenases by adding some cholinergic agonist or some Ca++ ionophore. I would think in a simple experiment just titrating the respiration with FCCP. This should increase the cytosolic ATP demand that would force the cells to speed the glycolysis and because it will uncouple the mitos it will increase the respiration as well. You could try to compare the uncouple respiration after adding oligomycin in muscle fibers in the presence and absence of glucose and pyruvate. I would think that this procedure may maximize the chances of you seen a difference in the rates of O2 consumption.

@@ Line 1: / Line 1: @@
 __TOC__
-== Application in [[HRR]] (updated instructions) ==
-:::'''Glc:  Glucose''' (D-(+)-Glucose,  C<sub>6</sub>H<sub>12</sub>0<sub>6</sub>);  Sigma G 8270, 100 g, store at RT; M = 180.16 g·mol<sup>-1</sup>
+== Former Instructions ==
-:::: 2021-02: The preparation instructions were updated to take the volume of the solute (Gp) into account. The concentrations prepared following the former instructions (see Discussion section) are sufficiently high for SUIT protocol titrations.
-::::'''Preparation of 2 M stock solution''' (1 mL, dissolved in H<sub>2</sub>O):
+These instructions were given until 2020-03-08. They were updated because the volume of the solute was not taken into account. Therefore the concentration oft the stock solution was lower than intended.
+::::'''Preparation of 2 M stock solution''' (dissolved in H<sub>2</sub>O):
 ::::# weigh 360.32 mg of Glucose
-::::# dissolve in 775 µL H<sub>2</sub>O in 1.5 mL Eppendorf
+::::# dissolve 1 mL H<sub>2</sub>O in 1.5 mL Eppendorf
-:::»'''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
+::::'''Preparation of 2.2 M stock solution''' (dissolved in H<sub>2</sub>O):
-::::* Titration volume ('''2-mL O2k-chamber'''): 20 µL using a 50 µL Hamilton syringe.
+::::# weigh 79.2 mg of Glucose
-::::* Titration volume ('''0.5-mL O2k-chamber'''): 5 µL using a 25 µL Hamilton syringe.
+::::# dissolve 0.2 mL H<sub>2</sub>O in 0.5 mL Eppendorf
-::::* Final concentration: 20 mM
-::::'''Preparation of 2.2 M stock solution''' (200 µL, dissolved in H<sub>2</sub>O):
-::::# weigh 79.2 mg of Glucose
-::::# dissolve 155 µL H<sub>2</sub>O in 0.5 mL Eppendorf
-:::»'''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
-::::* Titration volume ('''2-mL O2k-chamber'''): 5 µL using a 25 µL Hamilton syringe.
-::::* Final concentration: 5.5 mM (1 g/L)
-::::* Titration volume ('''2-mL O2k-chamber'''): 10 µL using a 25 µL Hamilton syringe.
-::::* Final concentration: 11 mM (2 g/L)
 == MiPNet discussion forum: glucose oxidation in skeletal muscle (2013-10-29) ==