Preclinical exploration of combined glucagon inhibition and liver-preferential insulin for treatment of diabetes using in vitro assays and rat and mouse models.

AIMS/HYPOTHESIS: Normalisation of blood glucose in individuals with diabetes is recommended to reduce development of diabetic complications. However, risk of severe hypoglycaemia with intensive insulin therapy is a major obstacle that prevents many individuals with diabetes from obtaining the recommended reduction in HbA1c. Inhibition of glucagon receptor signalling and liver-preferential insulin action have been shown individually to have beneficial effects in preclinical models and individuals with diabetes (i.e. improved glycaemic control), but also have effects that are potential safety risks (i.e. alpha cell hyperplasia in response to glucagon receptor antagonists and increased levels of liver triacylglycerols and plasma alanine aminotransferase activity in response to glucagon receptor antagonists and liver-preferential insulin). We hypothesised that a combination of glucagon inhibition and liver-preferential insulin action in a dual-acting molecule would widen the therapeutic window. By correcting two pathogenic mechanisms (dysregulated glucagon signalling and non-physiological distribution of conventional insulin administered s.c.), we hypothesised that lower doses of each component would be required to obtain sufficient reduction of hyperglycaemia, and that the undesirable effects that have previously been observed for monotreatment with glucagon antagonists and liver-preferential insulin could be avoided. METHODS: A dual-acting glucagon receptor inhibitor and liver-preferential insulin molecule was designed and tested in rodent models (normal rats, rats with streptozotocin-induced hyperglycaemia, db/db mice and mice with diet-induced obesity and streptozotocin-induced hyperglycaemia), allowing detailed characterisation of the pharmacokinetic and pharmacodynamic properties of the dual-acting molecule and relevant control compounds, as well as exploration of how the dual-acting molecule influenced glucagon-induced recovery and spontaneous recovery from acute hypoglycaemia. RESULTS: This molecule normalised blood glucose in diabetic models, and was markedly less prone to induce hypoglycaemia than conventional insulin treatment (approximately 4.6-fold less potent under hypoglycaemic conditions than under normoglycaemic conditions). However, compared to treatment with conventional long-acting insulin, this dual-acting molecule also increased triacylglycerol levels in the liver (approximately 60%), plasma alanine aminotransferase levels (approximately twofold) and alpha cell mass (approximately twofold). CONCLUSIONS/INTERPRETATION: While the dual-acting glucagon receptor inhibitor and liver-preferential insulin molecule showed markedly improved regulation of blood glucose, effects that are potential safety concerns persisted in the pharmacologically relevant dose range.

Characterization of nerolidol biotransformation based on indirect on-line estimation of biomass concentration and physiological state in batch cultures of Aspergillus niger.

Biotransformation of the sesquiterpenoid trans-nerolidol by Aspergillus niger has previously been investigated as a method for the formation of 12-hydroxy-trans-nerolidol, a precursor in the synthesis of the industrially interesting flavor alpha-sinensal. We characterized biotransformations of cis-nerolidol, trans-nerolidol, and a commercially available cis/trans-nerolidol mixture in repeated batch cultures of A. niger grown in computer-controlled bioreactors. On-line quantification of titrant addition in pH control allowed characterization of (1) maximal specific growth rate in exponential growth phases, (2) exponential induction of acid formation in postexponential phases, (3) inhibition of organic acid formation after nerolidol addition, and (4) exponential recovery from this inhibition. Addition of a (+/-)-cis/trans-nerolidol mixture during exponential or postexponential phase to cultures grown in minimal medium at high dissolved oxygen tension (above 50% air saturation), to cultures at low dissolved oxygen tension (5% air saturation), or to cultures grown in rich medium demonstrated that the physiological state before nerolidol addition had a major influence on biotransformation. The maximal molar yield of 12-hydroxy-trans-nerolidol (9%) was obtained by addition of a (+/-)-cis/trans-nerolidol mixture to the culture in the postexponential phase at high dissolved oxygen tension in minimal medium. Similar yields were obtained in rich medium, where the rate of biotransformation was doubled.

Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter.

This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K(s)) of a reference strain was about 15 microM in glucose-limited chemostat culture. Disruption of mstA resulted in a two- to fivefold reduction in affinity for glucose and led to expression of a low-affinity glucose transport gene, mstC, at high dilution rate. The effect of mstA disruption was more subtle at low and intermediate dilution rates, pointing to some degree of functional redundancy in the high-affinity uptake system of A. niger. The mstA disruptant and a reference strain were cultivated in glucose-limited chemostat cultures at low, intermediate and high dilution rate (D=0.07 h(-1), 0.14 h(-1) and 0.20 h(-1)). Mycelium harvested from steady-state cultures was subjected to glucose uptake assays, and analysed for expression of mstA and two other transporter genes, mstC and mstF. The capacity for glucose uptake (v(max)) of both strains was significantly reduced at low dilution rate. The glucose uptake assays revealed complex uptake kinetics. This impeded accurate determination of maximum specific uptake rates (v(max)) and apparent affinity constants ( ) at intermediate and high dilution rate. Two high-affinity glucose transporter genes, mstA and mstF, were expressed at all three dilution rates in chemostat cultures, in contrast to batch culture, where only mstC was expressed. Expression patterns of the three transporter genes suggested differential regulation and functionality of their products.

Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production.

Chemostat cultivation of Aspergillus niger and other filamentous fungi is often hindered by the spontaneous appearance of morphologic mutants. Using the Variomixing bioreactor and applying different chemostat conditions we tried to optimize morphologic stability in both ammonium- and glucose-limited cultures. In most cultivations mutants with fluffy (aconidial) morphology became dominant. From an ammonium-limited culture, a fluffy mutant was isolated and genetically characterized using the parasexual cycle. The mutant contained a single morphological mutation, causing an increased colony radial growth rate. The fluffy mutant was subjected to transformation and finally conidiospores from a forced heterokaryon were shown to be a proper inoculum for fluffy strain cultivation.