Transient postweaning expression of excessive fat deposition and diabetes mellitus in ob/ob mice.

Growth, glucose levels and body composition of male and female C57BL/6 ob/ob and lean (?/+) mice were examined between 17 days and 1 year of age. Obese mice displayed three distinct periods of growth with each phase characterized by a different pattern of glucoregulation. Until 4 weeks of age, ob/ob mice had slightly higher rates of weight gain and fat accretion than littermate lean mice. Glycemia was in the normal range and insulin levels were elevated. Shortly after the transition to solid food, ob/ob mice displayed several weeks of increased body growth and very high rates of fat deposition. During this period of rapid weight gain, hyperglycemia arose despite progressively increasing IRI concentrations. Examination of individual obese mice at this time revealed that glycemia was highly correlated with the rate of body fat deposition. The final phase of the ob/ob syndrome began at 3 months of age, as rates of fat deposition abruptly slowed to values slightly less than those of lean mice and glucose values declined to normal although hyperinsulinemia and glucose intolerance persisted. The present results show that ob/ob mice, although characterized throughout life by many metabolic and growth disturbances, only display diabetes mellitus and exaggerated fat deposition during a 6 to 8 week period just after weaning.

Effects of epinephrine on thermoregulatory behavior in lean and obese Zucker rats in the cold.

This series of experiments examined whether epinephrine (EPI) produces the same thermoregulatory effects in the cold that have been reported for norepinephrine and isoproterenol. Lean and obese Zucker rats were trained to press a lever to activate infrared heat lamps in a cold (-8 degrees C) environment. Operant thermoregulatory behavior increased dose-dependently following EPI (0-100 micrograms/kg), but posttest colonic temperature (Tc) fell. Thermal balance calculations showed a substantial increase in net heat loss, more so in obese than lean animals. EPI is therefore thermolytic--i.e., disrupts thermal balance. A low dose (100 micrograms/kg) of the alpha-antagonist phentolamine produced a marked improvement in operant behavior, Tc, and thermal balance, whereas a comparable dose of the beta-antagonist propranolol had no beneficial effect. Increasing the dose of phentolamine worsened the responses with respect to the 100-micrograms/kg dose. The selective alpha 1-antagonist prazosin ameliorated the decrease in Tc induced by EPI but had little effect on operant behavior or thermal balance; the selective alpha 2-antagonist yohimbine had no effect on any parameter compared to EPI alone. These results suggest that the paradoxical effects of EPI in the cold are mediated by alpha-adrenoceptors, but definitive identification of the subclass of receptor involved cannot be determined.

Effects of beta-adrenoceptor agonists and antagonists on thermoregulation in the cold in lean and obese Zucker rats.

This experiment examines whether the thermoregulatory ability of obese Zucker rats is comparable to that of lean rats following treatment with beta-adrenoceptor agonists and antagonists in a cold (-8 degrees C) environment. Half-maximal doses of the nonselective beta-adrenoceptor agonist isoproterenol (ISO) produced net thermolytic (heat loss) effects in both obese and lean rats in an operant lever pressing for radiant heat task. ISO increased the demand for heat, but posttest colonic temperature (Tc) decreased. A low dose of propranolol (100 micrograms/kg) normalized thermoregulatory behavior, Tc, and thermal balance when coadministered with ISO. Activation of thermogenesis with the selective beta 3-agonist BRL 35135 (BRL) reduced heat influx by both obese and lean rats at doses between 2 and 10 micrograms/kg, but no dose-response effects were evident within this range. Posttest Tc and thermal balance indicated no thermolytic effects. No evidence was found for a beta 2-component in the BRL response when a supramaximal dose (40 micrograms/kg) was tested with the selective beta 2-antagonist ICI 118551 (1 mg/kg). These data show that, despite a higher baseline demand for heat, the obese Zucker rat responds to the thermogenic effects of BRL and the thermolytic effects of ISO as does the lean rat.

Sex differences in glycated hemoglobin in diabetic and non-diabetic C57BL/6 mice.

Pathophysiological implications of gender may be important in a number of disease states. We therefore decided to study the influence of gender on glycation in mice. Plasma glucose and glycated hemoglobin levels were determined by ion exchange (HbA1c) and/or affinity chromatography (GHb) in C57BL/6 ob/ob mice during the onset and subsequent decline of hyperglycemia. In preweanling ob/ob mice, glucose and glycated hemoglobin concentrations were equal to those of lean sex-matched controls. Shortly after weaning, plasma glucose in ob/ob mice increased to reach a maximum between 2 and 3 months of age, then declined over the next several weeks to levels within the range of lean mice. HbA1c values were closely associated with the glycemic changes. Male mice of both phenotypes consistently had higher values of glycated hemoglobin at a given glucose value than did females. Disappearance rates of chromium-labeled erythrocytes were slightly higher in lean female mice than in other subgroups but after correcting for phenotype/sex differences in blood volume, no phenotype or gender differences in RBC lifespan were observed. We conclude that there are gender differences in glycation of hemoglobin in mice and that factors other than RBC turnover are associated with the gender effects in both obese and lean mice.

Interactions between insulin and glucocorticoids in the maintenance of genetic obesity.

Glucoregulation and body composition were examined in 3-mo-old C57BL/6 ob/ob mice 6 wk after streptozotocin (STZ) or STZ plus adrenalectomy. STZ depressed somatic growth in ob/ob mice but did not cause hyperglycemia until immunoreactive insulin (IRI) was 40% (100 microU/ml) that of intact ob/ob mice. When IRI approached that of lean mice (40 microU/ml), ob/ob mice displayed severe hyperglycemia (800+ mg/dl) and other sequelae of type I diabetes but still maintained the same 50% body fat as untreated obese mice. In contrast, STZ diabetes in lean mice caused disproportionate reductions in body fat. Adrenalectomy before STZ led to the same insulinopenia, depressed growth, and hyperglycemia as STZ alone, but, after combined treatment, percent body fat declined in proportion to IRI. Thus a subgroup of severely diabetic adrenalectomized STZ obese mice with very low IRI (20 microU/ml) had body fat contents and fat-free masses equal to those of weight-matched lean mice. The data suggest that hypercorticoidism rather than hyperinsulinemia is largely responsible for obesity in ob/ob mice. However, in the absence of adrenal glucocorticoids, or perhaps with just their normalization, hyperinsulinemia appears necessary for maintaining excessive body energy stores.

Effects of phenotype, feeding condition and cold exposure on thyrotropin and thyroid hormones of obese and lean mice.

The effects of acute cold exposure on rectal temperature (Tr) and circulating thyrotropin (TSH), thyroxine (T4) and triiodothyronine (T3) levels were examined in fed and food-deprived obese (ob/ob) and lean (?/+) C57BL/6 mice. At 23 degrees C, obese mice had lower body temperatures but higher TSH, T4 and T3 values than lean mice while male mice of both phenotypes had similar body temperatures and higher levels of all three hormones than females. Obese mice became severely hypothermic during 4 h cold exposure (8 degrees C) although TSH and T4 concentrations declined equally in obese and lean mice and T3 values were unaffected by cold. Male and female mice exhibited similar Tr responses to cold, while males continued to have higher values of TSH and thyroid hormones than females. When allowed food during cold exposure, both obese and lean mice displayed higher Tr although obese mice remained hypothermic. Thyroid hormones in all groups were increased by feeding but only male mice exhibited increased TSH values. These data show that the acute feeding, metabolic and thermogenic responses of mice to low ambient temperatures are not closely associated with altered systemic levels of TSH, T4 or T3. Furthermore, since ob/ob mice did not display lower hormone levels or defective hormone responses to cold or feeding, the data suggest that their apparent hypothyroidism is largely independent of hormone availability to target tissues.

Ineffectiveness of parenteral fluoxetine or RU-486 to alter long-term food intake, body weight or body composition of genetically obese mice.

The effects of 3 weeks of daily subcutaneous injections of a serotonin agonist (fluoxetine, 20 mg/kg) or agent having antiglucocorticoid properties (RU-486, 20 mg/kg) on food intake and body growth were examined in C57BL/6 ob/ob and lean mice. Fluoxetine injections during ad libitum feeding led to depressed food intake and body weight in both obese and lean mice over the initial 10 days of treatment with full recovery of both intake and weight by the end of the study. Fluoxetine treatment of mice restricted to 3.2 g of laboratory diet per day caused small but persistent depressions of body weight in both phenotypes. RU-486 did not affect food intake weight or weight gain in either phenotype or feeding condition and neither drug affected carcass composition. Insulin levels were also unaffected by treatment but final corticosterone concentrations were consistently higher in fluoxetine-treated mice and lower in RU-486-treated mice. Although transient benefits on feeding and weight gain were observed with fluoxetine administration, the present results show that neither fluoxetine nor RU-486 administered parenterally provides significant long-term improvement in the metabolic, growth or behavioral disturbances that characterize ob/ob mice.

Thyroid hormone responses to feeding in ob/ob mice.

Many of the disturbances which characterize adult C57BL/6 ob/ob mice, including obesity, hypometabolism and hypothermia could arise from reduced circulating levels of thyrotropin and thyroid hormones. In the present study, measurement of these hormones in ad libitum-fed obese and lean mice housed at 22 degrees C revealed that mutant mice had levels of TSH equal to those of their ?/+ siblings, while total T4 and T3 concentrations were slightly higher than those of lean controls. The hormonal responses of obese mice to overnight food deprivation or to meal ingestion were also similar to those of lean control mice. Males of both phenotypes typically had higher TSH, T4 and T3 concentrations than did females, and in male mice the circulating levels of each hormone were much more responsive to the feeding condition. The present data are consistent with recent reports of defective target tissue responses and impaired hormone deiodination rather than depressed pituitary-thyroid hormone levels in accounting for the metabolic disturbances which characterize ob/ob mice.

Food restriction normalizes somatic growth and diabetes in adrenalectomized ob/ob mice.

Carcass composition was determined in intact and adrenalectomized (ADX) ob/ob mice after 6 wk of food restriction from weaning (21 days). Diets (2.6 or 3.2 g/day) led to reduced weight gain in intact ob/ob mice, but fat deposition was still greater, and lean growth was less than isocalorically fed lean mice. When diet was combined with ADX, obese mice fed 3.2 g/day had rates of fat-free body growth equal to ad libitum-fed lean mice but still gained more weight and deposited more fat. With 2.6 g/day, however, body weight gain, fat deposition, fat-free body growth, and protein deposition were all similar to intact lean mice receiving the same diet. Neither diet nor diet combined with ADX improved the defective thermoregulation of obese mice tested at 23 degrees C, 8 degrees C, or in response to food deprivation or food ingestion. Together, diet and adrenalectomy reduced blood glucose and insulin levels in ob/ob mice, and with the 2.6 g/day diet, the values of each were the same as littermate lean controls. The present results indicate that adrenal glucocorticoids are necessary for maintaining elevated fat deposition at the expense of lean body growth in dieted ob/ob mice, whereas hyperphagia, defective thermoregulation, energy efficiency, and diabetes are still expressed after surgery.

Hormonal responses during development of streptozotocin diabetes in rats.

The pancreatic B-cytolytic agent streptozotocin (STZ) was administered at high and low doses to male Sprague-Dawley rats at the time of weaning (20-30 d). During the next 15 weeks growth, food intake, glycemia and circulating insulin (IRI), glucagon (IRG) and corticosterone (CS) levels were measured at regular intervals. Within seven days after receiving the highest dose of STZ the rats exhibited a severe and persistent diabetes characterized by depressed growth, elevated food intake, insulinopenia and increased concentrations of IRG and CS. With lower doses, a slowly developing hyperglycemia was observed which after a delay of 4 to 6 weeks equalled in severity that of the high dose group. There were also corresponding delays in the appearance of the other major symptoms of the diabetes. Glucose tolerance tests performed 6 weeks after STZ showed that both high and low dose groups were severely glucose intolerant and neither group exhibited an IRI response to glucose challenge. Multiple regression analyses of the hormone levels of individual rats during the developing diabetes revealed that hyperglucagonemia and hyperglycemia arose simultaneously, while insulinopenia was delayed in appearance and only correlated significantly to glycemia late in the experiment. These data suggest that streptozotocin diabetes initially arises from increased glucagon production coupled with a limited capacity of the pancreas to secrete insulin in response to hyperglycemia. Subsequently, the contribution of other factors including continued B-cytolysis, insulinopenia, and elevated adrenal cortical hormones results in the development of the full diabetic syndrome.

Adrenalectomy reduces but does not reverse obesity in ob/ob mice.

Bilateral adrenalectomy (ADX) of 4-month-old ob/ob mice led to reduced rates of body weight gain, a complete cessation of fat deposition and increased percentage carcass protein and ash during a 2-month observation period after surgery. However, ADX obese mice were still heavier and had more body fat and lower concentrations of carcass protein and ash than intact sex-matched littermate lean mice at the end of the experiment. When adrenalectomy was performed in younger obese mice before the syndrome was fully expressed (23 +/- 2 days of age), body weight gain was reduced by 40 per cent and fat deposition by 50 per cent during the next 3.5 months, but each was still greater than that of littermate lean mice. Despite the lower rate of weight gain after adrenalectomy, the skeletal and lean body growth of the early ADX obese mice equalled that of both obese and lean mice fed ad libitum. When the carcass composition of early ADX obese mice was compared with that of intact obese mice which were calorically restricted to the same rate of body weight gain, the ADX group had significantly less carcass fat (28 per cent) and more protein (50 per cent) and ash (20 per cent) than the dieted obese mice. In both experiments adrenalectomy led to reduced circulating immunoreactive insulin levels, although hyperinsulinemia persisted. The present results show that adrenalectomy is an effective tool for ameliorating the severity of many aspects of the ob/ob syndrome, particularly when compared with caloric restriction, but the procedure does not entirely reverse the deranged metabolism or abnormal carcass composition of these mice.

Effects of estrogen on food intake, body weight, and temperature of male and female obese mice.

Estradiol benzoate (EB) treatment of male and female C57BL/6J ob/ob mice for 32 days led to decreased body weight (20%), percentage body fat (8%) and carcass protein content (12%) when compared with non-EB-treated obese control mice. Estradiol reduced the caloric intakes of both genders by 25-35%, but did not affect body temperature regulation. Circulating glucose and insulin concentrations were also lowered by estrogens, although hyperinsulinemia persisted. Since post-treatment body weight changes correlated with daily food intakes (r = 0.81) rather than to rectal temperatures (r = -0.19), it appears that hypophagia provided a greater contribution to the estrogen-mediated reductions of growth and carcass fat than did altered energy expenditure for thermoregulation. While these data show that EB treatment does reduce the severity of some metabolic disturbances in a genetic model of type II diabetes, long-term estrogens do not appear to offer substantial advantages in the treatment of obesity or diabetes when compared with the effects of caloric restriction alone.

Fed and fasting thermoregulation in ob/ob mice.

The body temperatures of mature lean and obese C57BL/6J mice were measured just after feeding, during ad libitum access to food, or every 24 h throughout a 3-day fast. Obese mice had body temperatures 1.0-1.4 degrees C lower than lean mice in the postprandial state and during ad libitum feeding. During food deprivation, obese mice became more hypothermic than lean sex-matched controls. A 5 degrees C fall in body temperature was observed in mutant females in the first 24 h of starvation, about twice that seen in any other experimental group. Over the same period the temperature changes of obese males and lean females were similar and both groups had larger hypothermic responses than lean males. The present results indicate that both genotype and gender affect thermoregulation in these mice. Under normal colony room conditions (ad libitum feeding, 23 degrees C) the ob/ob mutation is expressed by lower body temperatures which along with hypoactivity and hyperphagia account for the high rates of energy storage. When food availability is limited, females of both phenotypes display an increased capacity to reduce their maintenance energy requirements by lowering body temperatures. This hypothermia may be responsible for both the increased conservation of body mass seen during starvation and the slightly greater (5%) fat stores observed in female mice.

Temperature preference of genetically obese (ob/ob) mice.

Genetically obese (ob/ob) and lean mice selected their preferred ambient temperature in a thermal gradient. Preferred ambient temperature was defined as that ambient temperature which the mice selected for sleep during daylight hours. Lean mice selected a temperature of 31.2 degrees C which resulted in a body temperature (36.7 degrees C) not greatly different from the pretest body temperature of 36.4 degrees C. Obese mice selected 29.4 degrees C which resulted in a body temperature of 36.8 degrees C, 1.8 degrees C above the pretest body temperature of 35.0 degrees C. These data indicate that obese mice select an ambient temperature that results in a body temperature no different from that of lean mice. The selection by obese animals of an ambient temperature significantly lower than that of lean mice but which results in the same body temperature may reflect an effect of adiposity on heat loss. There is no evidence of a diminished thermoregulatory set-point in obese mice.

The effects of exercise and food restriction on obesity and diabetes in young ob/ob mice.

Hormone levels and body composition were examined in six-week-old C57BL/6J ob/ob mice following 25 d of limited caloric intake, voluntary exercise, or combined treatment. Pair-feeding obese mice to the daily intakes of lean mice reduced body weight gain, skeletal growth and lean body mass. Although weight gain was the same in the two phenotypes, ob/ob mice had fourfold higher rates of fat deposition. When exercise was combined with pair-feeding, skeletal and lean body growth were reduced even further and weight gain was now less than ad libitum-fed lean controls. Carcass fat accretion, however, continued to be two to three times greater. No single treatment reversed the hyperglycemia or elevated hormone production of obese mice, although slightly lower values of glucose, insulin, glucagon and corticosterone were associated with pair-feeding. When diet was combined with exercise, fasting glycemia and glucagonemia were reduced to equal the values of lean mice but insulin and corticosterone levels remained elevated. The present results show that dieted and exercised ob/ob mice continue to exhibit very high rates of fat deposition even though skeletal and lean growth are severely limited. Since fat accretion is maintained under these conditions, it appears that obese mice are not just storing excess calories as fat, but are actively regulating body fat content to levels about 30 percent higher than lean mice.

Glucose tolerance in aging obese (ob/ob) and lean mice.

Examination of the glucose tolerance in younger (3 month) and older (6 month) obese mice revealed that most of their postinjection hyperglycemia arises from the disproportionately large glucose responses to the injection/bleeding procedures rather than from the added glucose. Simultaneous measurements of circulating glucagon, corticosterone and insulin indicated that simple differences in the levels of these hormones, in their circulating ratios, or in the magnitude of the hormone responses to stimulation did not fully account for the "stress"-induced hyperglycemia.

Starvation and age effects on glycoregulation and hormone levels of C57BL/6Job/ob mice.

The contributions of insulin (IRI), glucagon (IRG), and corticosterone production to the glycemic changes associated with age and starvation were examined in 3 and 6 month old ob/ob and lean mice. Three month old ob/ob mice had elevated glucose levels under all feeding conditions, but in older obese mice basal hyperglycemia was evident only after 48 hours of food deprivation. These age differences in glycoregulation were not consistently related to changes of IRI, IRG, or corticosterone concentrations. Similarly, the mild diabetes associated with senescence in lean mice was only evident during food deprivation. This abnormality in glycoregulation was also independent of decreased IRI or elevated diabetogenic hormone concentrations. Our results indicate that there is no simple hormonal basis for the partial remission of diabetes in older ob/ob mice, or for the development of mild diabetes in aging lean mice. Additionally, these data suggest that the tissue 'resistance' that is associated with chronic insulin overproduction might also develop in response to persistent overproduction of other metabolically-active hormones.

Glucose and endocrine responses to hypothalamic electrical stimulation in rats.

The effect of ventromedial (VMN) or basolateral (LHA) hypothalamic electrical stimulation on glucose and hormone production was examined in conscious and anesthetized male rats. Barbiturate treatment alone led to large increases of corticosterone and smaller but significant increases of glucose, insulin, and glucagon. When hypothalamic stimulation was combined with anesthesia, interactions between the hypothalamic sites and the awake-anesthetized conditions were observed. This was most evident with LHA placements, as awake rats exhibited mild hypoglycemia and hypoglucagonemia in response to low-level (approximately 10 microA rms) current, but no effects on these values were seen with VMN electrodes. With barbiturate anesthesia, the same level of stimulation led to severe hyperglycemia in both VMN and LHA rats and a mild hyperglucagonemia in the LHA group. No consistent effects of stimulation on immunoreactive insulin or corticosterone were detected at either hypothalamic site or in any anesthesia condition. The present results point out the important role anesthetics may play in studies defining functional aspects of the hypothalamus. Furthermore, our inability to elicit consistent autonomic responses with electrical stimulation, when compared with the effectiveness of both metabolic agents and pharmacologic stimuli, suggests that the sites integrating autonomic function may be diffusely distributed over the basal diencephalon.