A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice.

AIMS/HYPOTHESIS: C57BL/6J mice exhibit impaired glucose tolerance. The aims of this study were to map the genetic loci underlying this phenotype, to further characterise the physiological defects and to identify candidate genes. METHODS: Glucose tolerance was measured in an intraperitoneal glucose tolerance test and genetic determinants mapped in an F2 intercross. Insulin sensitivity was measured by injecting insulin and following glucose disposal from the plasma. To measure beta cell function, insulin secretion and electrophysiological studies were carried out on isolated islets. Candidate genes were investigated by sequencing and quantitative RNA analysis. RESULTS: C57BL/6J mice showed normal insulin sensitivity and impaired insulin secretion. In beta cells, glucose did not stimulate a rise in intracellular calcium and its ability to close KATP channels was impaired. We identified three genetic loci responsible for the impaired glucose tolerance. Nicotinamide nucleotide transhydrogenase (Nnt) lies within one locus and is a nuclear-encoded mitochondrial proton pump. Expression of Nnt is more than sevenfold and fivefold lower respectively in C57BL/6J liver and islets. There is a missense mutation in exon 1 and a multi-exon deletion in the C57BL/6J gene. Glucokinase lies within the Gluchos2 locus and shows reduced enzyme activity in liver. CONCLUSIONS/INTERPRETATION: The C57BL/6J mouse strain exhibits plasma glucose intolerance reminiscent of human type 2 diabetes. Our data suggest a defect in beta cell glucose metabolism that results in reduced electrical activity and insulin secretion. We have identified three loci that are responsible for the inherited impaired plasma glucose tolerance and identified a novel candidate gene for contribution to glucose intolerance through reduced beta cell activity.

Dietary fibers and heavy metal retention in the rat.

The metal-binding capacities of some gel-forming polysaccharides and other substances have been investigated in vitro in an attempt to relate their metal-binding properties to the retention of dietary Pb and Cd in vivo. In equilibrium dialysis systems, aqueous solutions of alginic acid, pectin, agar, and carrageenan (1 g fiber/100 ml) all bound Pb and Cd to varying degrees. Alginic acid had the greatest binding capacity for Pb (50 micrograms Pb bound/mg fiber) and carrageenan for Cd (9.3 micrograms Cd bound/mg fiber). Addition of any one of these fibers, or indulin or glucuronic acid to the diet increased the tissue retention of one or both of the metals. Only cellulose supplementation reduced the retention of both Pb and Cd. Carrageenan decreased that of Pb and increased that of Cd. In another experiment alginic acid was shown to increase Pb retention in rats even when present at fairly low dietary concentrations (1 g/kg).

Metal absorption and the intestinal mucus layer.

The luminal mucus layer of the rat's small intestine was separated from the mucosa by filling the lumen with agar jelly. When the rats were fasted overnight and given 59Fe or 65Zn by stomach tube the mucus isolated by this technique contained more isotope and more sialic acid than fed controls, and more of the isotope was absorbed into the tissues. Saturation and time-course studies showed that the mucus layer has metal-binding activity distinct from that of the mucosa and it is postulated that the mucus layer may play a role in the process of metal absorption.

The relationship between iron status and lead absorption in rats.

The absorption of lead from loops of small intestinein situ was investigated in rats in which iron absorption was increased by stimuli varying in type, intensity, or duration. Lead absorption was increased by a short period of severe iron restriction before any change in hematological indices became apparent. A period of hypoxia, which markedly increased iron absorption, did not influence absorption of lead. An extended period of moderate iron restriction resulted in a marked reduction in liver iron stores and increased iron absorption throughout the 17-wk experiment. Under these conditions lead absorption was initially also increased, but after 12 wk, when iron intake had become adequate to meet essential requirements, lead absorption was similar to that in iron-supplemented rats. These results are discussed in the light of evidence for a receptor-mediated absorption process for iron.

Effects of dietary phytic acid on lead and cadmium uptake and depletion in rats.

Rats were given a semipurified diet supplemented with phytate (10 g/kg) or calcium (6 g/kg) and lead (200 mg/kg) or cadmium (5 mg/kg) for 4 weeks. Addition of phytate or calcium reduced the accumulation of lead in bone (P less than 0.001) and in blood and liver samples (P greater than 0.05). The greatest inhibition of tissue lead retention was evident when phytate and calcium were fed together. Cadmium accumulation was measured in the liver and kidney and was increased (P less than 0.05) by the addition of calcium. Phytate inhibited the increase in tissue cadmium promoted by calcium supplementation but did not otherwise influence tissue cadmium levels. In a further experiment, weanling rats were given diets supplemented with cadmium (5 mg/kg) or lead (200 mg/kg) for 4 weeks, and the accumulation of these elements in the body tissues was estimated in some animals. A phytate-supplemented (10 g/kg) or phytate-free semipurified diet (free of lead and cadmium) was then given to the remaining rats for 4 weeks. Phytate supplementation was found to have no significant effect on the rate of loss of lead or cadmium from tissues.

The production of zinc deficiency in the guinea pig.

A diet which contained less than 1 microgram Zn per g was eaten readily by guinea pigs. No signs of zinc deficiency were produced unless the animals were deprived of access to zinc-supplemented solid food from birth and were maintained on grid floors to prevent coprophagy or ingestion of sawdust bedding material. At an early stage of deficiency, plasma zinc concentration, food intake and growth were reduced and large regular variations of food intake (food cycling) were observed. More severe deficiency produced decreases in the zinc content of other tissues and characteristic epidermal lesions. The requirement of guinea pigs for zinc for growth is probably about 2.5 micrograms per g feed which is much lower than for calves, rats and pigs.

The effects of short periods of fasting on the absorption of heavy metals.

1. Rats were deprived of food for periods of from 0 to 40 h and then given 203Pb, 203Hg, 59Fe, 64Cu, 65Zn or 45Ca by stomach tube. The absorption and retention of these metals in tissues was measured 1 h and 2 d after dosing. 2. After 16-24 h of food deprivation between two and ten times more metal was retained than after 0-12 h. 3. The effects of length of fast on metal uptake by the intestinal mucosa were greatest in the duodenum. 4. Lactate production by duodenal mucosa was halved after 12 h of food deprivation but galactose absorption was little changed after a 40 h fast.

The influence of high dietary calcium and phosphate on lead uptake and release.

The effects of calcium and phosphate supplements on the retention of dietary lead and the loss of lead from the body were studied in young rats. There were three experiments in which rats were given a diet containing 200 or 400 micrograms of lead/g for 3 or 6 weeks. The diet contained adequate calcium and phosphate. Calcium and phosphate supplements were given alone or together. Lead uptake after a period of lead feeding was measured by analysis of the whole gut-free carcass. Lead loss was measured by a similar analysis after the rats had received diets containing lead and then diets free of lead. The uptake of lead from the diet was reduced by about half when either dietary calcium or phosphate or both was doubled. The rate of release of body lead was decreased by calcium supplementation.

Dietary protein and lead retention.

The effects of dietary protein content on lead retention were studied in young rats. In experiments lasting between 3 and 6 weeks, rats given diets containing lead and 20% casein were fed ad libitum or were pair fed with rats given diets containing 6.5 or 7.5% casein. Among rats given 20% casein, lead retention was inversely related to growth rate. A reduction of dietary protein either had no effect or reduced lead retention. In other experiments lasting 4 days and using 203Pb, given orally or intraperitoneally, the recovery of isotope in the gutfree carcass was unaffected or reduced by a reduction in dietary protein. Blood and liver lead often increased when kidney and carcass lead decreased in response to a reduction of dietary protein. Conflicting observations on the effect of low-protein diets on lead retention may thus be due to opposing effects of low dietary protein and decreases of growth rate and to different responses of different tissues.

The effect of age on the absorption and excretion of lead.

The retention (the proportion of ingested or administered lead found in the carcass at slaughter) of lead in the carcass and tissues of rats given 203Pb intraperitoneally or by mouth was measured over a period of a few days at 3-month intervals for 9 months from weaning. Total carcass retention of 203Pb administered intraperitoneally reached a minimum at 6 months of age, but the retention of 203Pb in blood, liver, and kidney changed little with time. When 203Pb was given by mouth, the fraction of the dose absorbed and the fraction retained in the carcass reached a minimum at 6 months, but the recovery in the tissues continued to decrease up to the end of the experiment. The addition of 20 mg of lead/kg of diet had no consistent effect on the metabolism of 203Pb.