The effects of growth hormone derived polypeptides on the metabolism of isolated epididymal adipose tissue

1. Acid hydrolysis of growth hormone yields two polypeptides which have been called Ac-G (Acceleratory fraction, growth hormone} and In-G (Inhibitory fraction, growth hormone}. 2. Previous studies in this laboratory have shown that In-G inhibited glucose metabolism in isolated skeletal muscle, fatty acid synthesis in liver slices and extracts, and the oxidation of pyruvate in skeletal muscle and liver slices. These effects were localized to the inhibition of the triose phosphate dehydrogenases, acetyl CoA carboxylase and pyruvate dehydrogenase respectively. Ac-G abolished all of these effects by reversing the inhibition of each of these enzymes by In-G. 3. The studies described in this thesis were concerned with the possibility that the lipolytic effect of growth hormone may be mediated through the actions of these polypeptides. Lipolysis was studied using rat epididymal fat pads, isolated fat cells and cell-free extracts. 4. Initial experiments established that In-G inhibited the intermediary metabolism of adipose tissue at the same sites previously shown to be affected in liver and muscle; i.e., the triose phosphate dehydrogenases, acetyl CoA carboxylase and pyruvate dehydrogenase. 5. Following the development of a technique for labelling the glycerol fractions in fat pads, the effects of Ac-G and In-G on the release of labelled glycerol from the tissue triglycerides was investigated. In-G stimulated lipolysis; Ac-G alone had no effect, but abolished the lipolytic effect of In-G; insulin was ineffective in this regard. 6. Subsequent experiments studied the lipolytic activity of cell-free fractions of adipose tissue prepared essentially by the methods of Shafrir and Gorin (1964) and Chlouverakis (1968). Neither Ac-G nor In-G affected lipolytic activity in these preparations. 7. This suggested that the stimulation previously observed in whole fat pads might be mediated by some indirect effect of the polypeptide. Two such mechanisms were considered - (1) that the polypeptide might increase the synthesis of the intracellular lipases; or (2) that In-G might stimulate lipolysis as a result of one of its previously established metabolic effects. 8. The lipolytic effect of In-G was unaffected by cycloheximide. This ruled out the possibility that an increased de novo synthesis of the lipases might be involved. 9. Consequently, some explanation for the lipolytic effect of ln-G was sought in terms of its inhibition of the triose phosphate dehydrogenases with subsequent alteration to the concentrations of glycolytic intermediates. Accordingly, the various glycolytic intermediates were tested for their influence on lipolytic activity in cell-free preparations. FDP, which stimulated lipolytic activity by more than 100%, was the only one of these intermediates which had any effect whatsoever. Similar observations were also reported by Chlouverakis (1968). l0. It was found that when In-G stimulated lipolysis, the levels of FDP in whole fat pads and adipocytes were increased by more than 400%. Ac-G reversed both of these effects. 11. From the experiments described above, the following mechanism for the lipolytic effect of In-G was proposed: (i) that In-G inhibited the triose phosphate dehydrogenases; (ii) that the inhibition of these enzymes led to an increase in the tissue FDP concentration; (iii) that these increased FDP levels, in turn, stimulated the relevant intracellular lipases, perhaps by some form of allosteric activation as proposed by Chlouverakis(1968). 12. However, the concentration of FDP required to stimulate lipolytic activity in cell-free extracts was approximately 102 times higher than the average concentrations determined in fat pads and isolated cells treated with In-G. Nevertheless, this disparity was not considered to be inconsistent with the concept outlined above, since such differences in effector concentrations may simply reflect fundamental differences between the integrated metabolism of the whole cell and the activity of cell-free extracts. 13. When fat pads vvere treated with In-G, elevated levels of FDP were observed within 12 minutes. No effect on glycerol release was detected at this point; only subsequently was there any increase in the rate of glycerol release. This indication that stimulation of FDP levels preceded stimulation of lipolysis was consistent with the proposed causal relationship between FDP concentrations and rates of lipolysis. 14. From this it appears very likely that the lipolytic effect of growth hormone may be explained in terms of the actions of In-G as detailed in paragraph 11 above.