Inability of muscles in the obese mouse (ob/ob) to respond to changes in body weight and activity.

The fat-free carcass weight of the obese mouse (ob/ob) is generally less than that of wild-type siblings. The aim of this investigation was to examine the effect of obesity on muscle weights and histochemistry and to determine whether any effects could be eliminated when the obesity was largely prevented or reduced by limiting food intake. For 5 muscles examined the weights were significantly greater (except for biceps brachii) in the wild-type than in obese mice. Although there was a significant correlation between muscle weight (except for soleus) and body weight in the wild-type mice, no such correlation held for the obese mice. No remarkable differences between groups of mice were found in the histochemistry of the biceps brachii and soleus muscles except that fibre sizes were generally smaller in the obese mice. It is concluded that the skeletal muscles of obese mice cannot respond to the increased activity associated with prevented or reduced obesity.

Increased neuropeptide-Y messenger ribonucleic acid (mRNA) and decreased neurotensin mRNA in the hypothalamus of the obese (ob/ob) mouse.

The obese hyperglycaemic ob/ob mouse exhibits hyperphagia and other abnormalities of hypothalamic function. We measured hypothalamic concentrations of four peptides implicated in the control of appetite and energy expenditure, neuropeptide-Y (NPY), neurotensin, galanin, and somatostatin, by RIA and their respective mRNAs using semiquantitative Northern blotting. Using lean (+/+) mice as controls, we found unchanged concentrations of NPY, galanin, and somatostatin and a 25% reduction in neurotensin (P < 0.01). Neurotensin mRNA was similarly decreased (by 30%; P < 0.02), while NPY mRNA was increased 3-fold (P < 0.01). Centrally administered neurotensin decreases food intake, whereas NPY potently stimulates food intake. An increase in NPY gene expression together with reductions in neurotensin concentration and mRNA in the hypothalamus may be implicated in the development of hyperphagia and other neuroendocrine abnormalities seen in the ob/ob mouse.

Abnormal incisor teeth and body weight in the obese mouse (genotype ob/ob).

Inherently obese mice (genotype ob/ob) developed abnormal incisor teeth at 26 weeks of age. Up to that age, their teeth were indistinguishable by visual criteria, from those of lean (wild-type) litter-mate mice. Radiography and preliminary histology suggested impaction of the tooth in its alveolus (socket) due to the disorganized production of enamel and dentine. Incidence was high (92 per cent) in obese and zero in lean mice. Upper incisor teeth were more severely affected than lower. The severity of teeth lesions could not be correlated with age or body weight. Both sexes were equally affected. The onset of teeth lesions marked the end to the rapid rise in body weight characteristic of the obese mouse. An irregular fall in body weight ensued which could be alleviated by powdering the pelleted food. This indicated the fall to be a consequence of impaired function of the incisor teeth. Abnormality of the teeth was entirely prevented by feeding obese mice from weaning, a similar amount of food to that eaten by lean mice. The high circulating levels of adrenocorticosteroids in the obese mouse are suggested as a cause of the incisor tooth abnormality.

Measurement of peptidoleukotrienes in biological fluids.

Samples of human bronchoalveolar lavage fluid (BALF) and urine were utilized to demonstrate methods for quantitation and validation of leukotrienes (LTs). These methods utilize an enzyme immunoassay (EIA) that uses commercially available reagents, the antibody recognizing LTC4, LTD4, LTE4, and N-acetyl LTE4. BALF containing epithelial lining fluid was collected from atopic asthmatics both before and 5 min after the subjects had been challenged with a local instillation of allergen into the airways. BALF samples collected without allergen challenge had low levels of immunoreactive LTs, whereas samples collected after allergen were markedly elevated. After high-performance liquid chromatography (HPLC) separation of LTs, EIA revealed the presence of LTC4. The identity was validated by incubating LTC4 with a bovine gamma-glutamyl transpeptidase with dipeptidase activity that converted added [3H]-LTC4 as well as LTC4 immunoreactivity to LTE4. Urine samples collected from six healthy volunteers, one patient with adult respiratory distress syndrome (ARDS), and three patients in status asthmaticus were also analyzed for LTs. After HPLC separation of LTs and quantitation by EIA, urine samples from healthy subjects were found to have low but measurable LTE4. In contrast, the urine samples from the patients in status asthmaticus and from the ARDS patient had large elevations of LTE4 levels compared with healthy subjects. When the HPLC fractions containing [3H]LTE4 and LT immunoreactivity in the ARDS sample were treated with acetic anhydride, HPLC analysis indicated that both radiolabel and immunoreactivity now eluted at the retention time of N-acetyl LTE4, the derivatized product of LTE4. The methods described are relatively easy and can be used to measure and validate the existence of peptidoleukotrienes in biological samples.

The effect of limit feeding on thermogenesis and thermoregulation in genetically obese (ob/ob) mice during cold exposure.

Thermogenesis and thermoregulation in ad-lib-fed and limit-fed lean (+/ob or +/+) and obese (ob/ob) mice during acute cold exposure were studied by measuring oxygen consumption and body temperature. No significant differences in oxygen consumption were found between the lean ad-lib, obese ad-lib- or obese limit-fed groups. The oxygen consumption of the lean-limit-fed group was decreased by 25-30 per cent compared with the other groups. The body temperature of the obese ad-lib-fed group fell at a rate of at least twice that of any other group. The weight, total cytochrome c oxidase activity and protein content of the brown adipose tissue (BAT) of the lean groups was similar, and there appeared to be little difference in cell size or fat content. The BAT of both obese groups showed a several-fold increase in weight, and a 50 per cent increase in total protein, compared with the lean groups. The limit-fed obese group showed a significant increase in cytochrome c oxidase activity compared with all other groups. The BAT cells of both obese groups were much enlarged and contained considerable amounts of fat. These observations indicate that the susceptibility of obese mice to hypothermia is not due to a reduced capacity for thermogenesis, but to a failure to conserve heat. Failure of thermoregulation in obese animals may be due to postural constraints that result in increased heat loss by radiation. The results are discussed in relation to the accredited role of BAT thermogenesis in rodents exposed to the cold.

The role of L3T4+ cells in the pathogenesis of lupus in lpr-bearing mice. I. Defects in the production of interleukins 2 and 3.

Mice which bear the lpr gene spontaneously develop autoimmune syndromes characterized by massive expansion of an unusual T cell subset which is phenotypically Thy-1+, L3T4-, Lyt-2-, B220+. The mutant T cells are refractory to stimulation with mitogenic lectins and, by implication, are thought to be solely responsible for the defects in lymphokine production manifested by lpr mice. The contribution of the remaining L3T4+ T cell subset to the latter derangements has not been previously examined and is the focus of this study. We found that abnormalities in concanavalin A-induced interleukin 2 and 3 production in the spleens of MRL-lpr/lpr and C57BL/6.lpr mice occurred in the presence of limited infiltration with B220+, L3T4- T cells. Mixing experiments indicated that B220+ T cells were not suppressive. Furthermore, lpr spleen cells enriched for L3T4+ cells and depleted of sIg+, B220+ and Lyt-2+ cells demonstrated reductions in lymphokine production which were comparable to those seen in unfractionated preparations. Spleen cells from C57BL/6.lpr mice, enriched for L3T4+ cells, were also markedly impaired in a mixed leukocyte reaction in response to stimulator cells from the class II major histocompatibility complex mutant bm12. The results indicate that the aberrations in lymphokine production and proliferation in the spleen cells of lpr mice involve not only B220+ T cells but also L3T4+ cells and suggest a potential role for the L3T4+ subset in the pathogenesis of lupus in lpr-bearing mice.

Influence of restricted food intake on brown adipose tissue function in genetically obese mice (genotype, ob/ob).

Measurements were made of cytochrome c oxidase activity and the GDP-binding capacity of mitochondria in brown adipose tissue of genetically obese mice and wild-type siblings, to estimate the thermogenic capacity of the tissue. The binding capacity was decreased in ad libitum fed obese animals compared with wild-type animals. Limited feeding of obese animals to restrict their body weight caused a large increase in the binding capacity of the tissue, which was greater than that in wild-type animals fed either ad limitum or on a limited diet. The decreased binding capacity of brown adipose tissue mitochondria in obese mice appears to be a consequence of ad libitum feeding and therefore not a cause of the obesity. Limit feeding of obese animals also corrected their characteristic hypothermia at low ambient temperature. The large increase in the thermogenic capacity of brown adipose tissue in obese animals, induced by limited feeding, may account for the vital improvement of their thermoregulation. However, close similarities were found between obesity hypothermia and hypothermia induced in wild-type animals by restraint. It is suggested that changes in posture caused by obesity, resulting in increased loss of body heat, may be important in the development of obesity hypothermia. Obese animals fed less than wild-type grained more weight than wild-type animals, indicating that the high thermogenic capacity of their brown adipose tissue did not function to regulate their calorie intake.

Decreased food intake in response to cholecystokinin (pancreozymin) in wild-type and obese mice (genotype ob/ob).

Cholecystokinin decreased food intake more effectively in obese mice than in wild-type mice. Following a 23-h fast and 10 min after an ip injection of 0, 30, 60 or 90 U/kg. CCK in physiological saline, eating, drinking and rearing rates were measured for a period of 20 min. Thirty units affected neither group, 60 U slowed the eating rate of obese mice significantly, and 90 U that of both groups, particularly the obese mice (P less than 0.001). Drinking and rearing rates remained unchanged. Obese mice were not hyperphagic under the conditions of the experiment and showed an increase in the latency period which preceded eating, compared with wild-type mice. The enhanced responsiveness to CCK, both in and out of the hyperphagic state, may be associated with low endogenous levels of satiety hormones in the obese mouse.

Investigation into the hypogonadism of the obese mouse (genotype ob/ob).

Features of the reproductive axis in the genetically hypogonadal, obese mouse (genotype, ob/ob) were examined at 5-8 months of age and compared with those of wild-type litter mates. Hypothalamic concentrations of dopamine and 5-hydroxytryptamine were normal. Those of 5-hydroxyindoleacetic acid, noradrenaline and LH-RH were raised. LH-RH was biologically active. Pituitary concentration of LH was normal, but that of FSH was raised. Serum concentrations of LH and FSH, compared with those of wild-type animals, were normal and low, respectively. Gonad and accessory sex organs weights were reduced. These findings suggest that the release of FSH but not LH is defective in the ob/ob mouse. Preliminary in-vitro experiments indicated that the pituitary gland responded normally or even supernormally towards LH-RH in its release of LH. The defect in the reproductive axis of the obese mouse may be due to inadequate release of LH-RH although an insensitivity of the pituitary gland towards LH-RH in its release of FSH cannot be excluded.

Development of the hypothermia in obese mice (genotype ob/ob).

Heavier-obese mice (genotype, ob/ob) displayed their characteristic hypothermia at ambient temperatures and obese mice at all body weights did so after 60 min in the cold (3.0 - 5.5 degrees C). The fall in core temperature was least in slightly obese (3.5 degrees C) and greatest in extremely-obese animals (12.5 degrees C). The fall could be prevented by previous pair feeding with wild-type animals, which limited the body weight by 52 per cent. Limiting the body weight of wild-type mice (23 per cent) was not followed by a change in core temperature. Neither grouping nor floor insulation altered the fall in core temperature of obese or wild-type mice. These results suggest the hypothermia of the mutant to be as much under a physical as a metabolic influence, such as the abnormal body form and consequent inactivity. Heat production in muscle would decrease with the inactivity, contributing to the hypothermia. The normothermia in obese mice of limited body weight could result from their increased (normal) activity, previously demonstrated.

Acute effects of nicotine on plasma free fatty acid concentrations and on the response to cold stress, in lean and obese (genotype ob/ob) mice.

Lean and genetically obese (ob/ob) male and female mice were given nicotine by subcutaneous injection. Nicotine treatment was found to raise plasma free fatty acids by similar amounts in both lean and obese mice. In lean mice, nicotine caused depression of rectal temperature at ambient temperatures 22-25 degrees C and partially prevented the hypothermia in these mice when exposed to cold (o-3 degrees C). In obese mice, nicotine treatment did not alter either rectal temperature at 22-25 degrees C or the severe hypothermia on cold exposure. It is proposed that the effect of nicotine on free fatty acids is due to release of adrenal catecholamines and that this mechanism operates in both lean and obese animals. It is also proposed that, in obese mice under normal circumstances, there is a defect in the central nervous control of this adrenergic mechanism which may contribute to the observed fall in body temperature at low ambient temperatures.

Abnormal dentition and decrease in body weight in the genetically obese mouse (genotype, ob/ob).

Loss of body weight accompanied by cannibalism occurred in obese mice (genotype, ob/ob) at seven months of age. The onset was sudden. The animals had accompanying abnormalities of their incisor teeth. Some upper incisor teeth had no erupted part. The unerupted part of all the incisor teeth showed ridged thickening. Body weight loss was prevented by grinding the pelleted food to a powder. Possible causes of the teeth abnormalities are discussed.

Potentiation of hyperphagia and relief of hypothermia in the genetically obese mouse (genotype, ob/ob) by alpha-methyl tyrosine.

DL-alpha-methyl-p-tyrosine methyl ester hydrochloride affected the hyperphagia and hypothermia characteristic of the genetically obese mouse (genotype, ob/ob) throughout an experimental period of 5 days. Intraperitoneal injections of 100 mg/kg body weight, daily, resulted in a significant increase in the average daily food consumption by 60 per cent, already elevated 35 per cent above that of lean litter-mates. The drug, administered at the same dose, caused a similar percentage elevation of food intake in the lean litter-mates. Rectal temperatures of obese mice were raised significantly throughout the 5-day period by an average of 0.95 degrees C, following administration of the drug. There was a significant rise of 0.75 degrees C in the rectal temperature of lean mice on 2 of the 5 days in the period. Body weight remained unchanged. Further experiments are necessary to determine the site of action at which DL-alpha-methyl-p-tyrosine brings about these effects at this dose in lean and obese mice.

Reproductive hormonal function in the genetically obese (ob/ob) mouse.

Reproductive function is impaired in the genetically obese (C57 B1/6J) ob/ob mouse. Serum LH, FSH, and testosterone concentrations were assessed in male ob/ob and lean littermates from 39 to 78 days of age. The lean animals demonstrated a three-fold rise in serum LH between 39 and 45 days of age that preceded a steep increase in serum testosterone which peaked at age 70 days. The obese animals did not demonstrate this LH rise; serum testosterone levels were low and had a blunted increase with age that paralleled that of normal animals. Serum FSH was lower than normal at all ages in the obese mice. The ventral prostrate and testes were small in the ob/ob mice. The castration of adult animals resulted in increased serum concentrations of both LH and FSH, with higher levels attained in the lean animals. Fifty-four-day-old castrated lean and obese mice were treated with testosterone for 15 days. Measurements of serum LH and FSH after 8 and 15 days of treatment demonstrated a marked sensitivity in the ob/ob animals to feedback inhibition of gonadotropins. This finding suggested persistent immaturity of the hypothalamic-pituitary axis in obese mice. These studies indicate that the hypogonadism of the ob/ob mouse is the result of altered hypothalamic-pituitary function.