A molecular investigation of the obese phenotype in the Aston University strain of ob/ob mice and the Japanese Kuo Kondo mice.

The aim of this work was to determine the role of the ob gene in the obese phenotype observed in the Aston University Strain of ob/ob mouse and the Japanese Kuo Kondo (KK) mouse. After RT-PCR amplification of the ob RNA, the transcript was cloned into the vector pCR3 and three individual clones from each strain were sequenced. It was confirmed that the Aston University strain of ob/ob mice shared the same C-T mutation found in the Jackson Laboratory C57BL/6J ob/ob strain whereas the Japanese KK mice showed wild-type ob gene expression. This study indicates that the ob mutation has survived unchanged following the separation of the two strains of ob/ob mice, and secondly, that the molecular basis of the obese phenotype in the KK mice is not due to mutations in the ob gene.

A re-evaluation of the anabolic effect of testosterone in rats: interactions with gonadectomy, adrenalectomy and hypophysectomy.

Growth and body composition were determined in rats implanted with silastic tubes containing testosterone. The implant (i) had no effect on growth in intact males, (ii) restored the sub-normal weight gain of gonadectomized males to normal (+30%; p < 0.01), (iii) substantially increased weight gain in intact females (+172%; p < 0.001), and (iv) caused a further increase (+32%; p < 0.01) in the accelerated weight gain resulting from ovariectomy. In intact animals of both sexes, testosterone caused significant atrophy of the reproductive tissues; this was accompanied in females by reduced plasma estradiol concentrations. Thus the large effect of testosterone on growth in intact females is probably due to diminished secretion of ovarian estradiol, and is distinct from the smaller effect observed in castrated animals of both sexes. To investigate the mechanism underlying the latter response, testosterone was implanted in rats which had been both adrenalectomized and gonadectomized, and also in hypophysectomized animals. In each case a significant anabolic effect was observed, showing that the response requires neither adrenal nor pituitary glands. In all experiments, increased body weights resulting from testosterone treatment consisted at least partly of fat-free mass.

The effects of fasting on plasma corticosterone kinetics in rats.

Plasma corticosterone clearance in anaesthetized rats was measured from the disappearance of radioactivity after a bolus injection of [3H]corticosterone. Mean fractional clearance rates were significantly (P less than 0.05) reduced after a 48 h fast, by 32 and 22% for males and females respectively. Plasma corticosterone concentrations were increased by fasting in both sexes. Corticosterone secretion rates, calculated as the product of fractional clearance and plasma corticosterone concentration, did not differ between fed and fasted groups in either sex. The mean activity (U/liver) of the rate-limiting enzyme for corticosterone degradation, hepatic 4,5-dihydrocorticosterone:NADP+ delta 4-oxidoreductase, was significantly reduced by 51 and 78% after fasting in males and females respectively. This was due to changes in both the soluble and microsomal forms of the enzyme. The binding capacity of corticosterone-binding globulin in plasma was significantly reduced by fasting in females (P less than 0.001), but was not altered in males. The results suggest that reduced hormone clearance is the dominant cause of fasting hypercorticosteronaemia in the rat.

Energy balance in rats given chronic hormone treatment. 2. Effects of corticosterone.

1. Sprague-Dawley rats were given corticosterone for 4 to 14 d either by subcutaneous injection (50 mg/kg body-weight per d) or as a higher dose in the diet (1 g/kg diet). Energy balance was calculated using the comparative carcass technique. 2. Corticosterone significantly suppressed growth rate by at least 50% (P less than 0.001 in all experiments). The reduction in growth was more marked in males than in females. 3. Hormone treatment significantly reduced metabolizable intake (kJ/d) in males but not in females. Expressed relative to either metabolic body size (kg body-weight0.75) or fat-free mass, metabolizable intake tended to be increased in the treated groups. 4. Energy expenditure, calculated as the difference between metabolizable intake and gain and expressed as kJ/d, did not differ between treated and control rats. Relative to either metabolic body size or fat-free mass, expenditure was consistently increased in treated rats. This change was statistically significant in five of the eight comparisons. 5. The corticosterone-treated rat is characterized by high energy intake and expenditure relative to its body size and growth rate. Alterations in the relative sizes of different lean tissues may contribute to these changes.

Energy balance in rats given chronic hormone treatment. 1. Effects of long-acting insulin.

1. Sprague-Dawley rats were injected for 16 d with long-acting insulin, and energy balance was calculated using the comparative carcass technique. Two experiments were carried out with females (starting weights 150 and 90 g respectively), and one with males (starting weight 150 g). In a fourth experiment, cytochrome c oxidase (EC 1.9.3.1) activity was measured as an indicator of the capacity for substrate oxidation. 2. Insulin increased weight gain by up to 57% (P less than 0.01 for all studies). Metabolizable energy intake (kJ/d) was also consistently higher in the treated groups, by up to 34% (P less than 0.01 for all studies). The excess weight gained by the insulin-treated rats was predominantly due to fat deposition. 3. Energy expenditure, calculated as the difference between metabolizable intake and carcass energy gain, was expressed on a whole-body basis, or relative to either metabolic body size (kg body-weight0.75) or fat-free mass. Insulin consistently raised energy expenditure, regardless of the method of expression, but this change reached statistical significance in only two of the nine comparisons. 4. Cytochrome c oxidase activity was not affected by insulin treatment in either interscapular brown adipose tissue or gastrocnemius muscle. In liver, total enzyme activity (U/tissue) was increased from 2928 (SE 162) in the controls to 3940 (SE 294) in the treated group (P less than 0.02), but specific activity (U/mg protein) was unchanged. 5. It is concluded that, despite causing substantial hyperphagia, insulin treatment only slightly increases energy expenditure in rats. The costs of increased tissue deposition may account for this change.

Digestibilities of casein and soya-bean protein in relation to their effects on serum cholesterol in rabbits.

Diets containing isolated soya-bean protein induce lower levels of serum cholesterol in animals than diets containing casein. Experiments were conducted to investigate whether differences in digestibility of the proteins might explain this effect. At pH 8 with pancreatic enzymes or intestinal peptidase, soya-bean protein was hydrolysed in vitro much less rapidly than casein. However, with pepsin (EC 3.4.23.1) at acid pH, soya-bean protein was hydrolysed more rapidly than casein. These differences in digestibility may be due to pH-dependent changes in solubility of the proteins. Casein and soya-bean protein were most soluble at alkaline and acid pH respectively. Heat treatment of the proteins resulted in lower solubilities and digestibilities. Sonication of soya-bean protein at pH 7.8 increased solubility but only slightly raised digestibility. When fed to rabbits, enzymically hydrolysed soya-bean protein induced a 2.3-fold higher concentration of serum cholesterol than did intact soya-bean protein. The hypocholesterolaemic effect of soya-bean protein may be partly attributable to its low solubility and digestibility at alkaline pH.

A longitudinal cross-over study of serum cholesterol and lipoproteins in rabbits fed on semi-purified diets containing either casein or soya-bean protein.

1. Two groups, each of six rabbits, were fed on semi-purified diets containing either 400 g casein or 400 g soya-bean protein/kg for 20 d and then the diets of the two groups were crossed-over. 2. Just before the cross-over, the serum cholesterol concentration (mean +/- SE) was 3068 +/- 592 and 800 +/- 143 mg/l for the groups fed on casein and soya-bean protein respectively. 3. Changes in the serum cholesterol concentration were observed 1 d after crossing-over the diets. By 10 d, the cholesterol levels in the two groups had also crossed-over. 4. The changes in serum cholesterol level after the cross-over were reflected in the very-low-density lipoproteins ( VLDL) and low-density lipoproteins (LDL). 5. Lipoprotein protein concentrations in the LDL changed in the same way as cholesterol. In the VLDL however, the protein concentration decreased in both groups after the change in diet. 6. The cholesterol: protein values for the LDL and VLDL markedly increased in the rabbits changed from the soya-bean-protein diet to the casein diet, reaching a maximum 2 d after the cross-over. In the animals switched from casein to soya-bean protein, the values progressively declined. 7. The source of dietary protein exerts a rapid effect on the composition of both the VLDL and LDL which is proposed to be attributed to changes in the number and size of lipoprotein particles.

The density profile and cholesterol concentration of serum lipoproteins in domestic and laboratory animals.

1. By means of density gradient ultracentrifugation, the density profile of the serum lipoproteins was studied in 14 species of domestic and laboratory animals: the pig, chicken, rhesus monkey, rabbit, dog, horse, sheep, cat, mouse, goat, cow, guinea-pig, trout and rat. 2. The concentration of cholesterol in whole serum and the lipoprotein fractions of these animal species was also determined. 3. There were large differences in the density profile of the serum lipoproteins among the various animals studied and the results indicate that the density limits employed for human serum lipoproteins are not directly applicable to the lipoproteins of animals. 4. In most of the animals studied, three major lipoprotein fractions could be observed as in man. 5. However, such a clear pattern was not found in the cow, rat and guinea-pig. 6. In the animals studied, with the exception of the pig and the guinea-pig, most of the cholesterol in the serum was carried in the high density lipoprotein fraction.

The rapid determination of carcass fat by the Foss-Let specific gravity technique.

1. Carcass fat was determined by extraction with tetrachloroethylene and measurement of the solvent's change in density. The results were comparable in precision to those of a reference method; the new method extracted storage lipid but little structural lipid. 2. The technique is simple, rapid and appropriate for many nutritional studies.

Calcitonin receptors of kidney and bone.

Receptors for calcitonin, determined by activation of adenylate cyclase, were found in a distribution among zones of the kidney distinct from that of receptors for parathyroid hormone or vasopressin. Competitive binding studies showed that the receptors for calcitonin are similar in kidney and bone and that their high apparent affinity for salmon calcitonin accounts in part for the high biological potency in vivo of salmon calcitonin.