In vitro and mouse in vivo characterization of the potent free fatty acid 1 receptor agonist TUG-469.

Activation of the G protein-coupled free fatty acid receptor 1 (FFA1; formerly known as GPR40) leads to an enhancement of glucose-stimulated insulin secretion from pancreatic ß-cells. TUG-469 has previously been reported as a potent FFA1 agonist. This study was performed to confirm the higher in vitro potency of TUG-469 compared to the reference FFA1 agonist GW9508 and to prove in vivo activity in a pre-diabetic mouse model. The in vitro pharmacology of TUG-469 was studied using Ca(2+)-, cAMP-, and impedance-based assays at recombinant FFA1 and free fatty acid receptor 4, formerly known as GPR120 (FFA4) expressing 1321N1 cells and the rat insulinoma cell line INS-1. Furthermore, we investigated the systemic effect of TUG-469 on glucose tolerance in pre-diabetic New Zealand obese (NZO) mice performing a glucose tolerance test after intraperitoneal administration of 5 mg/kg TUG-469. In comparison to GW9508, TUG-469 showed a 1.7- to 3.0-times higher potency in vitro at 1321N1 cells recombinantly expressing FFA1. Both compounds increased insulin secretion from rat insulinoma INS-1 cells. TUG-469 is > 200-fold selective for FFA1 over FFA4. Finally, a single dose of 5 mg/kg TUG-469 significantly improved glucose tolerance in pre-diabetic NZO mice. TUG-469 turned out as a promising candidate for further drug development of FFA1 agonists for treatment of type 2 diabetes mellitus.

NIDDM genes in mice: deleterious synergism by both parental genomes contributes to diabetogenic thresholds.

We used mouse genetics to model how polygenic thresholds for the transition from impaired glucose tolerance (IGT) to NIDDM are reached. NON/Lt and NZO/Hl are inbred mouse strains selected for IGT and polygenic obesity, respectively. Their F1 male progeny consistently developed NIDDM. Genetic analysis of F2 males from both cross directions identified an NON-derived diabetogenic locus, Nidd 1, on chromosome (Chr) 4 near the leptin receptor. This locus was associated with reduced plasma insulin, increased non-fasted blood glucose, and lower body weight. Another NON-derived diabetogenic locus on Chr 18 (Nidd2) that controls blood glucose was identified. An NZO-derived diabetogenic region on Chr 11 (Nidd3), possibly comprising two separate loci, reduced ability to sustain elevated plasma insulin and significantly reduced weight gain over time. Thus, the diabetogenic synergism between genetic loci from strains separately exhibiting subthreshold defects perturbing glucose homeostasis underscores the likely complexity of the inheritance of obesity-associated forms of NIDDM in humans.

Indomethacin treatment causes loss of insulin action in rats: involvement of prostaglandins in the mechanism of insulin action.

Glucose tolerance tests in rats showed that after indomethacin treatment plasma insulin levels rose five-fold higher than in untreated controls. Accordingly, the pancreatic islets of indomethacin-treated rats secreted insulin at a threefold higher rate. Glucose tolerance tests additionally showed that indomethacin treatment led to a retarded disposal of the elevated blood glucose. Both effects appear to be caused by an attenuation of the hormone responsiveness for insulin and noradrenaline (alpha-adrenoceptor action) by indomethacin. The following observations support this view: insulin and adrenaline (alpha-adrenoceptor action) lost their ability to lower cyclic adenosine monophosphate (AMP) levels in hepatocytes; the glycogen content of liver and skeletal muscle was reduced by 95% and 65%, respectively; in adipocytes the stimulation of glucose transport by insulin was reduced by 60%. These effects of indomethacin can be reversed by the addition of exogenous prostaglandin E (PGE), as elevated cyclic AMP synthesis was again sensitive to alpha-adrenergic inhibition in the liver. These results indicate a relationship between prostaglandins and insulin action. These effects of indomethacin could result from reduced synthesis of cyclic PIP (prostaglandylinositol cyclic phosphate), a proposed second messenger for insulin and alpha-adrenoceptor action, whose synthesis was decreased by indomethacin treatment and increased by the addition of exogenous PGE. Stimulation of glucose transport by cyclic PIP was unaffected by indomethacin treatment, in contrast to the stimulation by insulin. Inhibition of PGE and cyclic PIP synthesis resulted in a metabolic state comparable to insulin resistance in non-insulin-dependent diabetes mellitus.

Persistent MHV (mouse hepatitis virus) infection reduces the incidence of diabetes mellitus in non-obese diabetic mice.

The frequency of diabetes mellitus was compared in non-obese diabetic mice before and after inadvertent exposure of the colony to mouse hepatitis virus infection. Prior to exposure, diabetes prevalence and cumulative diabetes incidence in 7-month-old mice was 65% and 25% in females and males, respectively. Diabetes incidence/quarter revealed a seasonal pattern with peaks in winter. After mouse hepatitis exposure, the diabetes incidence in the colony decreased and testing for mouse hepatitis antibody in blood samples revealed a persistent infection. In the offspring of mice delivered by caesarean section, the diabetic incidence increased sharply from a nadir of 36% to 95% and from 9% to 65% in females and males, respectively. In individual mice, diabetes resistance was strongly correlated to high titres of mouse hepatitis virus antibody. The results of this inadvertent viral infection demonstrate that a diabetes-susceptible genotype is strongly modulated by environmental factors. Investigators studying this diabetes model should strive for specific pathogen-free colony status and a high incidence of diabetes before attempting to investigate therapeutic modalities.

Radiation induced formation of giant cells in Saccharomyces uvarum. IV. Macromolecular synthesis and protein patterns.

X-irradiated (1.0 kGy) yeast cells (Saccharomyces uvarum, ATCC 9080), grown in liquid medium stop their mitotic activities and form giant cells by development of several buds which do not separate from mother cells. Depending on the time in culture, wet and dry weights per cell, protein-RNA- and DNA- contents per cell as well as incorporation rates of 14C-leucine per cell and per hour and patterns (isoelectric focussing) of water soluble proteins were studied. Weights per cell, RNA and protein contents per cell and 14C-leucine incorporation rates increase markedly in giant cells, whereas DNA content per cell is only duplicated. Protein patterns in isoelectric focusing show one interesting difference. In samples from giant cells one protein band (IP = 6.63) decreases after 8 h in culture and later on disappears completely. This finding is not due to primary damage in X-irradiated DNA but seems to be related to the control of cell cycle events.

Giant cells from Saccharomyces uvarum grown after x-irradiation.

Yeast cells, Saccharomyces uvarum, were irradiated with X-rays and grown in liquid suspension. Glucose as the only carbon source was limited to 12.5 mM. Under these conditions giant cells are formed. Cell number, glucose utilization, ethanol production and oxygen consumption are measured during the time of growth. The mean weight of single cells in the stationary phase increases up to 75 krad and is not due to an uptake of water. In irradiated cultures oxygen consumption and glucose utilization per cell are higher than in control cells. The data demonstrate that synthesis- and energy-metabolism during the formation of non-dividing, radiation-induced giant cells is increased.