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Summary of full report
Gluconeogenesis in isolated equine
hepatocytes
by T. L. Frankel and M. Jois
RIRDC Web Publication No W04/113 RIRDC Project No ULA-14A
Executive Summary
This is the first report
of a method to isolate equine hepatocytes and measure gluconeogenesis in
vitro.
Glucose is an important energy source in equine athletes. However high concentrations of blood glucose has been suggested as a risk factor for bone disorders in young foals and for causing behavioural problems in adults. Starches in feed can be broken down in the small intestine to release glucose which can then be absorbed into the blood circulation. Glucose can also be synthesised from the volatile fatty acid (VFA) propionic acid which is produced by bacterial action on starches and non-starch polysaccharides that are not digested in the small intestine. It has been estimated that 57 to 68 % of glucose circulating in the blood of horses is derived from propionic acid but there is no information on the proportion of propionic acid that can be converted to glucose or on its rate of conversion.
The aim of the project was to develop a method to examine gluconeogenesis in equine liver cells and to determine the rate of glucose production from propionic acid and some of the factors that could influence the rate. The project also supported the Honours training of a student.
The variation in liver morphology, especially in the preferred caudate lobe, limited choice of a lobe with suitable in/out blood supply that could be used for isolation of hepatocytes. The left medial lobe was selected after examining a range of livers from different sized horses.
Using abattoir tissues rather than livers from purpose selected animals showed that sampling time after death and provision for the immediate perfusion of the lobe with an ice-cold EGTA solution was critical for the isolation of viable liver cells. Possible differences (between sheep and horses) in cell structure resulted in relatively long times of perfusion with greater amounts of collagenase solution.
Despite these changes a still unexplained ‘frothing’ occurred in the perfusion fluid and this had the potential to block small capillaries, reducing oxygen supply to cells and causing a reduction in cell viability.
If all conditions of isolation were optimal, viability of isolated cells was greater than 95%. Further adaptation from the method used in sheep, was needed before it was possible to demonstrate that viable equine hepatocytes were able to synthesis glucose from propionate at a rate of 0.01 to 0.05 nmol glucose/ mg dry cells/ min. The horse cells required additional nutrients to those required by sheep. The development of this method, especially using abattoir specimens, will allow for investigation of many aspects of liver function in horses not only the controls of glucose production.
