No abstract

Strategies for achieving global food security include identification of alternative feedstock for use as animal feed, to contribute towards efforts at increasing livestock farming. The presence of theobromine in cocoa pod husks, a major agro-waste in cocoa-producing countries, hinders its utilisation for this purpose. Cheap treatment of cocoa pod husks to remove theobromine would allow largescale beneficial use of the millions of metric tonnes generated annually. The aim of this study was to isolate theobromine-degrading filamentous fungi that could serve as bioremediation agents for detheobromination of cocoa pod husks. Filamentous fungi were screened for ability to degrade theobromine. The most promising isolates were characterized with respect to optimal environmental conditions for theobromine degradation. Secretion of theobromine-degrading enzymes by the isolates was investigated. Theobromine degradation was monitored by HPLC. Of fourteen theobromine-degrading isolates collected and identified by rDNA 5.8S and ITS sequences, seven belonged to Aspergillus spp. and six were Talaromyces spp. Based on the extent of theobromine utilization, four isolates; Aspergillus niger, Talaromyces verruculosus and two Talaromyces marneffei, showed the best potential for use as bioagents for detheobromination. First-time evidence was found of the use of xanthine oxidase and theobromine oxidase in degradation of a methylxanthine by fungal isolates. Metabolism of theobromine involved initial demethylation at position 7 to form 3-methylxanthine, or initial oxidation at position 8 to form 3,7-dimethyuric acid. All four isolates degraded theobromine beyond uric acid. The data suggest that the four isolates can be applied to substrates, such as cocoa pod husks, for elimination of theobromine.

The ob/ob mouse responds predictably to chronic treatment with large doses of pituitary GH with marked hyperglycemia and decreased glucose tolerance. The purpose of the present study was to characterize the metabolic alterations produced by GH that lead to this diabetogenic response in the ob/ob mouse in order to determine whether this animal might serve as a useful model for the study of the cellular mechanisms involved in the diabetogenic action of GH. Female ob/ob mice were treated sc for 3 days with either saline or 200 micrograms/day S-carboxymethylated human GH (RCM-hGH), a diabetogenic GH derivative lacking significant growth-promoting or insulin-like activities. Six hours before the start of the experiment, the animals were given a sc injection of 2 micrograms dexamethasone and deprived of food. RCM-hGH treatment produced marked increases in fasting blood glucose and plasma insulin concentrations, but had no effect on plasma glucagon or serum insulin-like growth factor I levels. It had no effect on liver glycogen level or in vitro hepatic glucose production in the absence or presence of pyruvate and lactate added to the incubation medium. By contrast, the in vitro stimulatory effects of insulin on [14C] glucose oxidation by isolated soleus muscle or segments of parametrial fat were greatly attenuated by RCM-hGH treatment, without changes in rates of basal glucose oxidation. This change in peripheral tissue responsiveness to insulin does not appear to involve glucose transport, since the in vitro stimulation by insulin of 3-O-[14C]methylglucose transport into isolated diaphragm muscle was not altered by RCM-hGH treatment. Moreover, the RCM-hGH-induced reduction in adipose tissue responsiveness to insulin does not appear to be mediated by a reduction in insulin binding, since [125I]iodoinsulin binding to adipocytes isolated from RCM-hGH-treated mice was similar to that to cells from saline-treated animals. Interestingly, the reduction in responsiveness to insulin seen with segments of adipose tissue from RCM-hGH-treated animals was not found with isolated adipocytes prepared from such tissue by collagenase digestion. These results suggest that the hyperglycemia and glucose intolerance produced in ob/ob mice by chronic GH treatment result primarily from increased peripheral tissue insulin resistance. Therefore, the ob/ob mouse provides a useful model to elucidate the cellular mechanism(s) of this aspect of the diabetogenic action of GH.

GH has acute stimulatory effects on amino acid transport and protein synthesis in a variety of tissues, but it has not been established whether these effects are expressions of the growth-promoting property of GH or of its separate insulin-like action. The 20,000-dalton structural variant of human GH (20K hGH) has been shown to have a high ratio of growth-promoting to insulin-like activity compared to native hGH (22K hGH), suggesting that it could be used as a tool to address the above question. Therefore, experiments were conducted to compare the relative abilities of native 22K hGH and 20K hGH, when added in vitro, to stimulate amino acid transport and protein synthesis in the isolated diaphragm of the female hypophysectomized rat. Paired intact hemidiaphragms were preincubated for 1 h in the absence or presence of various concentrations of 22K or 20K hGH. Then, 3-O-[14C]methylglucose was added to the medium to measure sugar transport as a test of insulin-like activity, and either alpha-[3H]aminoisobutyric acid acid or [3H] phenylalanine was also added to measure amino acid transport or protein synthesis, respectively, during a final hour of incubation. When the responses to the various concentrations of 22K and 20K were compared, 20K hGH was only about 20% as effective as 22K in stimulating 3-O-methylglucose transport, reflecting its markedly attenuated insulin-like activity on the diaphragm. Similarly, 20K hGH was only 20% as effective as 22K hGH in stimulating alpha-aminoisobutyric acid transport and phenylalanine incorporation into protein in the same muscles. Therefore, these findings support the idea that the rapid stimulatory effects of GH on amino acid transport and protein synthesis are expressions of the insulin-like action of GH and are not components of the response of target cells to its growth-promoting action.