In our previous studies, we found that the gluconeogenic genes (G6Pase and PEPCK) were down-regulated in KO rats compared with WT controls. Consequently, we performed the pyruvate tolerance test to examine the hepatic gluconeogenesis. However, when I injected 2 g/kg pyruvate (a stock solution of 20 % sodium pyruvate in sterile PBS, the pH of the solution was not adjusted because Na-pyruvate will not change the pH significantly) into SD rats, but they contorted their body into painful postures and 30 minutes later died. Actually, the concentration of pyruvate in my work is much higher than the physiological osmotic pressure of the rats. I also tried a lower concentration, but the change of blood glucose level was small. How could I solve this problem? Would anyone give me some suggestion or cues when performing this experiment?
Was the pH adjusted after dissolving Na-Pyruvate or did you happen to dissolve Pyruvic acid in PBS, pH 7.4?
Describe what is it you are trying to achieve.
Hi Tausif Alam! Thank you for your answer! I have modified my question as described above. Looking forward for you answer!
the amount of pyruvate may be too high therefore pyruvate can not changes to glucose via gluconeogenesis completely and part of it change to lactic acid and may causes acidosis. increased thiamine intake (B1) during increasing PA. It may help
If you already know that G6Pase and PEPCK activities are down-regulated in your KO animals compared to the wild type, I have to assume that you have already measured these activities in vitro in liver extracts. Is there any reason why that is not enough?
In an animal, under normal conditions, you are not very likely to see a great deal of increase in blood glucose levels because of the action of endocrine pancreas. Insulin released from beta cells will fairly quickly force the excess glucose into storage, making glucose level as a very imprecise readout.
Mr Tausif Alam, thank you for your critical thinking. Actually, we isolated RNA from liver tissue and performed the qRT-PCR and found that the gluconeogenic genes were down-regulated. On one hand, we could further study the upstream transcription factors that are responsible for gluconeogenesis (to know why the genes of the KO animal are changed). On the other hand, we could further study the consequence of this change. Because we did not find any significant differences in fasted blood glucose levels between KO and WT rats, we used the well-defined substrate of gluconeogenesis (that is pyruvate) as an stimulus to force the potential difference to come out. Does this idea make sense?
Mr Javad Pourreza, thank you for your answer! This really helps a lot! At first, we thought the concentration of the pyruvate was too high. once it entered the circulation and was not metabolized to glucose immediately, the red blood cells shrinked and formed thrombus in the blood capillary. And now, your answer highlights another important explanation. Perhaps we should try a dose of 1.5 g/kg. Many thanks!
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