Thermoregulation and non-shivering thermogenesis in the genetically obese (ob/ob) mouse

1. The capacity ofr thermoregulation and thermogenesis in lean and genetically obese (ob/ob) mice has been investigated. 2. At 4 degrees C ob/ob mice rapidly die of hypothermia, because of a reduced capacity for cold-induced thermogenesis, but the animals are able to survive if previously adapted to 12 degrees C. 3. At all environmental temperatures between 30 degrees C and 10 degrees C the body temperature of ob/ob mice is 2.0-2.5 degrees C below that of lean animals. This may be due to a lower "setting" for body temperature. 4. At 34 degrees C the oxygen consumption of obese mice is greater than that of the lean animals while at 30 degrees C it is similar. When the environmental temperature is below 30 degrees C the oxygen consumption of the lean mice is greater. The obese animals therefore expend less energy on thermoregulatory thermogenesis. 5. The capacity for non-shivering thermogenesis was measured in lean and obese mice by investigating the effect of an injection of L-nor-adrenaline (1000 microgram/kg body weight) on the metabolic rate at 31 degrees C. Non-shivering thermogenesis was reduced by one-half in the obese animals. 6. One cause of the obesity of the ob/ob mouse is its high metabolic efficiency. We suggest that this high metabolic efficiency is due, at least in part, to less energy being expended on thermoregulatory thermogenesis.     

Additive effects of leptin and topiramate in reducing fat deposition in lean and obese ob/ob mice

The objective of the present study was to investigate the effects of the antiepileptic drug topiramate (TPM) on components of energy balance in lean and obese (ob/ob) mice in the presence or absence of leptin. Lean and ob/ob mice infused with either leptin or phosphate-buffered saline were treated with TPM for 7 days. TPM was mixed into the diet and administered at a dose of 60 mg/kg/day, whereas leptin was infused at the rate of 100 microg/kg/day using osmotic minipumps, which were subcutaneously implanted in the interscapular region. Food intake and body weight were monitored throughout the study. Body composition was measured prior to and following treatment with TPM and leptin, using dual-energy X-ray absorptiometry (DEXA). Glucose (glucose oxidase method) and insulin (radioimmunoassay) were also determined. TPM and leptin significantly reduced body weight gain, food intake and body fat gain in obese mice. The effects of TPM and leptin on fat gain were also statistically significant in lean animals. There was no interaction of TPM and leptin on the energy balance variables, the effects of the two substances being additive instead. Leptin abrogated hyperinsulinemia in obese mutants whereas TPM did not alter insulin levels in either lean or obese mice. The combination of leptin and TPM led to the normalization of glucose levels in obese mice. Our study demonstrates an effect of TPM in leptin-deficient animals, which suggests that TPM does not require the presence of leptin to exert its effect. They also show that the effects of leptin and TPM can be additive. The treatment with leptin in ob/ob mice neither accentuated nor blunted the effect of TPM on energy balance.     

Y4 receptor knockout rescues fertility in ob/ob mice

Hypothalamic neuropeptide Y (NPY) has been implicated in the regulation of energy balance and reproduction, and chronically elevated NPY levels in the hypothalamus are associated with obesity and reduced reproductive function. However, it is not known which one of the five cloned Y receptors mediates these effects. Here we show that crossing the Y4 receptor knockout mouse (Y4(-/-)) onto the ob/ob background restores the reduced plasma testosterone levels of ob/ob mice as well as the reduced testis and seminal vesicle size and morphology to control values. Fertility in the sterile ob/ob mice was greatly improved by Y4 receptor deletion, with 100% of male and 50% of female Y4(-/-),ob/ob double knockout mice producing live offspring. Development of the mammary ducts and lobuloalveoli was significantly enhanced in pregnant Y4(-/-) and Y4(-/-),ob/ob females. Consistent with the improved fertility and enhanced mammary gland development, gonadotropin releasing hormone (GnRH) expression was significantly increased in Y4(-/-) and Y4(-/-),ob/ob animals. Y4(-/-) mice displayed lower body weight and reduced white adipose tissue mass accompanied by increased plasma levels of pancreatic polypeptide (PP). However, Y4 deficiency had no beneficial effects to reduce body weight or excessive adiposity of ob/ob mice. These data suggest that central Y4 receptor signaling specifically inhibits reproductive function under conditions of elevated central NPY-ergic tonus.     

Precursor-protein convertase 1 gene expression in the mouse hypothalamus: differential regulation by ob gene mutation,energy deficit and administration of leptin,and coexpression with prepro-orexin

The expression of precursor-protein convertase (PC)1, PC2 and paired basic amino acid cleaving enzyme four mRNA was studied by in situ hybridisation in regions of the hypothalamus involved in energy regulation in relation to obese (ob) gene mutation and energy deficit. PC1 gene was differentially expressed in hypothalamic nuclei of mice from different genetic backgrounds or energetic status, whereas no differences in expression were observed for either the PC2 or paired basic amino acid cleaving enzyme four genes. In obese ob/ob mice, PC1 mRNA levels were increased in the paraventricular nucleus, decreased in the lateral hypothalamus and unchanged in the ventromedial nucleus and arcuate nucleus relative to lean controls. In response to intraperitoneal injection of murine leptin, PC1 mRNA levels in obese ob/ob mice decreased in the arcuate nucleus, increased in the lateral hypothalamus and were unchanged in both the paraventricular nucleus and ventromedial nucleus. In mice deprived of food for 24 h, PC1 mRNA levels were reduced in the ventromedial nucleus, increased in the lateral hypothalamus and unchanged in the paraventricular nucleus and arcuate nucleus relative to ad libitum-fed controls. Overall, whilst the data show effects related to leptin and energetic status, they do not support a strong and consistent link between PC1 gene expression and energy balance. This suggests that if PC1 is important to the control of energy balance then protein expression and activity, rather than gene expression may be the more critical parameters of regulation. The relationship between PC1 and candidate energy balance-related genes in the lateral hypothalamus was investigated by dual in situ hybridisation. PC1 mRNA was localised in prepro-orexin mRNA expressing neurons in the lateral hypothalamus, which suggests a functional relationship.     

Does hyperinsulinemia in ob/ob mice cause an insulin-stimulated adipose tissue?

After a 1-h preincubation to remove endogenous insulin, adipose tissue of obese mice (C57BL/L4 ob/ob) had a lower rate of glucose metabolism than tissue which was not preincubated. In contrast, preincubation did not change the metabolism of adipose tissue from lean mice (C57B1/6J +/+). The preincubation effect was abolished in obese mice which had had their serum insulin levels lowered toward normal by streptozotocin treatment. Injection of anti-insulin serum to obese mice caused adipose tissue removed 15 min after the injection to display a rate of glucose metabolsim lower than that of tissue removed before the injection. No such effect was seen in lean mice. These data are consistent with the hypothesis that hyperinsulinemia in the obese mice causes a chronic state of insulin stimulation of their adipose tissue, possibly contributing to their high rates of lipogenesis and their obesity. Several lipogenic enzymes were measured in adipose tissue of both lean and obese mice, and no single enzymatic abnormality was detected which might explain the hyperlipogenesis. Pyruvate dehydrogenase and acetyl-CoA carboxylase were both insulin-sensitive enzymes in lean and obese mice.     

Nicotine suppresses energy storage through activation of sympathetic outflow to brown adipose tissue via corticotropin-releasing factor type 1 receptor

Nicotine is known to stimulate energy expenditure, although the precise mechanism is unclear. To clarify the involvement of corticotropin-releasing factor (CRF) in the mechanism by which nicotine increases energy expenditure, the effect of intraperitoneal injection of nicotine (0.1 or 0.5 mg/kg) on the release of noradrenaline (NA), a stimulator of thermogenesis, in brown adipose tissue (BAT) important for energy expenditure was examined in rats. We also examined the effects of CRF receptor subtype antagonists on the nicotine-induced change in BAT NA release. Nicotine significantly increased BAT NA release at a dose of 0.5 mg/kg, and the increase was completely blocked by antalarmin, a CRF type 1 receptor antagonist, but not by antisauvagine-30, a CRF type 2 receptor antagonist. These results suggest that nicotine increases energy expenditure by activating BAT function, and that CRF type 1 receptors are involved in the mechanism by which nicotine affects energy balance.     

Norepinephrine turnover in obese (ob/ob) mice: effects of age, fasting, and acute cold

The possibility that low sympathetic nervous system (SNS) activity in brown adipose tissue (BAT) of 8-wk-old obese (ob/ob) mice results from their gross obesity at that age was investigated. Norepinephrine (NE) turnover, an estimator of SNS activity, was measured in BAT and other organs of 2-wk-old preobese ob/ob mice, and at 4 and 8 wk of age. Rates of NE turnover were 36% slower in BAT of preobese ob/ob mice than in lean littermates and remained slow in their BAT at 4 (-66%) and 8 (-56%) wk of age. In heart, rates of NE turnover were 48% slower in preobese ob/ob mice than in lean littermates, but the difference diminished at 4 (-21%) and 8 (-16%) wk of age. Rates of NE turnover in white adipose tissue, liver, and pancreas of obese mice were generally comparable with rates in these organs of lean mice. Effects of fasting (24 h) and acute cold exposure (14 degrees C for 8 h) were also examined. In general, fasting lowered and cold exposure elevated NE turnover equally in obese and lean mice. Ob/ob mice housed at 23-25 degrees C exhibit low SNS activity in their BAT prior to the onset of gross obesity, even though SNS activity in their BAT responds normally to an acute cold stress. This low SNS activity probably contributes to their subsequent high efficiency of energy retention.     

Reduced norepinephrine turnover in brown adipose tissue of ob/ob mice

Obese (ob/ob) mice have a lower thermogenic capacity than lean mice. The possible role of brown adipose tissue (BAT) in this defect was investigated. Lean and obese mice were exposed to 33 (thermoneutral), 25, or 14 degrees C for up to 3 wk. BAT cytochrome oxidase activity and numbers of Na+-K+-ATPase enzyme units, enzymes involved in thermogenesis, were similar at 33 or 25 degrees C. Chronic exposure to 14 degrees C increased these enzymes 34 and 62%, respectively, in lean mice and nearly 150% in obese mice. Sympathetic nervous system activity, which stimulates thermogenesis in BAT, was evaluated by measuring norepinephrine (NE) turnover. At 25 degrees C, NE turnover rate in BAT of obese mice was only 40% as rapid as in BAT of lean mice. Chronic exposure to 33 degrees C depressed NE turnover in BAT of lean mice, but not in obese mice, whereas exposure to 14 degrees C accelerated NE turnover in both lean and obese mice. Lower sympathetic nervous system activity in BAT of obese mice at 25 degrees C is likely a major factor in their reduced nonshivering thermogenesis and resultant high efficiency of energy storage.     

Behavioural energy regulation in lean and genetically obese (ob/ob) mice

The role of behaviour in the control of energy regulation has been investigated in relation to environmental temperature, nutrition and genetics. Techniques of operant conditioning were used, with lean and genetically obese (ob/ob) mice being tested at three environmental temperatures (10, 20 and 30 degrees C) and on two feeding regimes (after a 24 hr fast and after feeding ad lib). They were allowed access to heat and food, although the design of the apparatus ensured that both were not available simultaneously. Both the lean and ob/ob showed an initial preference for heat when tested in a cold environment. At a low ambient temperature the ob/ob were dependent on the heater rather than food to increase rectal temperature, both when fasted and when fed. By contrast, the lean had a lower demand for heat than the obese and used the time to explore the environment and to feed. Food intake increased with an increase in ambient temperature in both genotypes. Possible reasons for this are discussed.     

Na+,K+-ATPase enzyme units in lean and obese (ob/ob) thyroxine-injected mice.

The possible involvement of Na+,K+-ATPase in the etiology of obesity in the obese (ob/ob) mouse was explored. The number of Na+,K+-ATPase enzyme units in skeletal muscle, liver, and kidneys from 4- and 8-wk-old obese and lean mice was estimated from saturable [3H]ouabain binding to particulate fractions. Neither phenotype nor age altered the Kd value for ouabain binding in these three tissue preparations. The total number of [3H]ouabain binding sites in hindlimb muscles was 35--55% lower in 4- and 8-wk-old obese mice than in their lean counterparts. However, the total number of [3H]ouabain binding sites in liver and kidneys of obese mice was similar to values observed in their lean counterparts. Because it has been suggested that ob/ob mice are hypothyroid, we investigated the response of Na+,K+-ATPase in these mice to thyroid hormone treatment (approximately 5 microgram thyroxine/day for 2 wk). The number of [3H]ouabain binding sites in the three tissues increased in both obese and lean mice injected with this relatively large dose of thyroxine, but the obese mice were 2--3 times more responsive than lean mice.     

Alprazolam reduces stress hyperglycemia in ob/ob mice

We have shown that the C57BL/6J ob/ob (obese) mouse, a commonly used model of type II diabetes mellitus, is not in fact consistently hyperglycemic except when exposed to environmental stress. In an attempt to modify stress hyperglycemia in this animal, we administered either a 5 mg/kg dose of the benzodiazepine alprazolam or vehicle (propylene glycol) intraperitoneally to both obese mice and their lean littermates prior to a rest and a stress period. Alprazolam modified the hyperglycemic effect of stress only in the obese mice. Alprazolam significantly reduced plasma corticosterone in obese animals at rest and following stress. In addition, alprazolam significantly increased plasma insulin in all animals at rest and following stress. These data suggest a possible role for benzodiazepines in the modification of stress hyperglycemia in type II diabetes mellitus.     

Behavioral response of the genetically obese (ob/ob) mouse to heat stress: effects of naloxone and prior exposure to immobilization stress

Genetically obese (ob/ob) mice, which possess abnormally elevated levels of pituitary β-endorphin and adrenocorticotropin exhibited less grooming, rearing and jumping during a five min exposure to different levels of heat stress compared to their lean littermate controls (ob/?). Naloxone had a diametrically opposite effect on rearing in these animals, particularly when exposed to low heat stress; it enhanced rearing in ob/ob mice and suppressed the rearing response in ob/? mice. Naloxone enhanced jumping in both the ob/ob mice and the ob/? mice. This effect was slightly, although not significantly, stronger in the obese mice. Finally, exposure to 10 min of immobilization stress before testing at 46°C, enhanced grooming and suppressed jumping in ob/ob and ob/? mice. Naloxone pretreatment reversed the effect of immobilization stress in ob/ob mice but not in their lean littermate controls. The data is discussed in terms of the differential involvement of pituitary endorphins in the behavioral response of ob/ob and ob/? mice to stress.     

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.     

Food intake during tumor growth: anorexia in genetically obese ob/ob mice and hyperphagia in lean mice

Recent findings indicate that obese (ob/ob) mice suffer a low incidence of lung metastasis and survive longer than lean (+/?) littermates following injection with B16 melanoma cells [34]. The present study examined the food intake of obese and lean mice during the growth of this tumor. Mice from both groups increased their food intake by small and approximately equal amounts during the first three quarters of the survival period following injection with 10⁶ cells, and body weights remained fairly stable. During the final quarter, however, obese mice became anorexic whereas lean mice became intensely hyperphagic; body weights changed accordingly. Thus, food intake is differentially affected by tumor growth in this form of genetic obesity.     

Increased severity of pentylenetetrazol induced seizures in leptin deficient ob/ob mice

Leptin modulates multiple ion channels making its net effect on brain excitability difficult to predict. One method of determining leptin's net effect on brain excitability is to examine brain excitability during chronic leptin deficiency. We compared the susceptibility of leptin deficient ob/ob and wild type mice to pentylenetetrazol (PTZ) induced seizures using continuous video electroencephalogram (EEG) recordings. We found that ob/ob mice were more likely to die and were more susceptible to generalized clonic and clonic-tonic seizures than wild type mice at submaximal PTZ doses. These findings suggest that chronic leptin deficiency in vivo increases seizure susceptibility.     

Opiate receptors, food intake and obesity

The present studies tested the effect of acute and chronic administration of naloxone on food intake of lean and genetically obese (ob/ob) mice. Acute administration of naloxone, a drug which blocks opiate receptors, produced a greater reduction of food intake in obese (ob/ob) mice than in the lean littermates. For chronic experiments with naloxone, the daily feeding period was shortened to eight hours and two injections of naloxone were given four hours apart. With this procedure of scheduled-feeding the food intake of both lean and obese mice was depressed during the first hour after injecting naloxone. However, beginning on the second day of treatment, the lean mice began to eat more food than the untreated controls during the eight hour feeding period. Food consumption by lean mice reached values 140 to 200% above the control levels between the fourth and sixth day. In the obese mice the rise in food intake was more gradual and did not reach 200% of the control value until the sixth day. Body weight changes reflected the changes in food intake. In contrast to naloxone, chronic treatment with morphine lowered food intake and blocked the stimulatory effect of naloxone. Our findings suggest that endogenous opioids may play a role in signalling satiety and in regulating long-term energy balance.     

Classically conditioned enhancement of hyperinsulinemia in the ob/ob mouse

The obese (C57BL/6J ob/ob) mouse is a commonly used animal model of non-insulin-dependent diabetes mellitus. Recent experiments have shown that stress hyperglycemia can be classically conditioned in the obese but not in the lean mouse. In the present study, classical conditioning of insulin secretion was attempted in C57BL/6J obese and lean animals. For 21 days, obese and lean mice were exposed to a conditioned olfactory stimulus prior to and during eating. On the 22nd day, blood was sampled for all animals following presentation of the conditioned stimulus; testing was repeated 2 weeks later following an additional 4 days of conditioning. Results indicated an effect of conditioning, with significantly greater plasma insulin for trained than for untrained obese mice. That insulin secretion can be more easily conditioned in the obese mouse suggests that a cholinergic mechanism may be involved in the hyperinsulinemia characteristic of this animal.     

Thermal preference behavior in preweaning genetically obese (ob/ob) and lean (+/?, +/+) mice

Impaired nonshivering thermogenesis and lowered rectal temperatures (Tre) are hallmarks that appear early in the postnatal ontogeny of the genetically obese (ob/ob) mouse. Adult obese mice compensate behaviorally for these impairments and do not defend their low Tres. We predicted that, because young mice primarily rely on behavior to ensure thermal homeostasis during preweaning development, the appearance of the obese mouse's thermoregulatory impairment should promote their continued reliance on behavioral thermoregulation compared to lean pups. Accordingly, intact litters of pups from heterozygous lean (C57BL/6J, ob/+) and from homozygous lean (+/+) matings were tested at 6, 12, and 18 days postpartum on a thermal gradient (14-44 degrees C). Obese pups had lower pretest Tres than lean (+/?) littermates at 6 days and lower pretest Tres than both lean littermates and homozygous (+/+) lean control pups at 12 and 18 days. Exposure to the gradient ameliorated these differences (i.e., no posttest Tre differences among phenotypes). Correspondingly, obese pups preferred warmer gradient locations than +/+ pups but similar locations to their phenotypically lean (+/?) littermates until 18 days, when both lean groups preferred similar thermal locations compared to warmer-seeking obese pups. These data support our hypothesis and emphasize the age-dependent impact of the ob gene on altering mouse pups' thermal preferences.     

Leptin regulates olfactory-mediated behavior in ob/ob mice

We have investigated olfactory-mediated pre-ingestive behavior in leptin (ob/ob) and leptin receptor (db/db) mutant mice compared to age- and gender-matched wild-type (wt) mice. Olfactory-mediated behavior was tested using a buried food paradigm 5 times/day at 2-h intervals for 6 days. Mean food-finding times of ob/ob and db/db mice were approximately 10 times shorter than those of wt mice. To test the effect of leptin replacement in ob/ob mice, leptin (1 or 5 microg/g body weight in sterile saline) or carrier was injected i.p. once daily prior to testing. Mean food finding times in ob/ob mice injected with carrier or with 1 microg/g leptin were similar and were 2-3 times faster than in wt mice. Mean food finding times in ob/ob mice injected with 5 microg/g leptin tripled compared to carrier-injected ob/ob mice and were of the same order of magnitude as those of wt mice, suggesting functional leptin replacement. A 3-factor repeated measures ANOVA demonstrated significant differences between the 6 cohorts (P = 0.0001), food finding times (P< or = 0.0001), and cohort by day interaction (P< or = 0.0001). Post hoc tests suggested that the ob/ob+5 mug/g leptin cohort performed more like the wt cohort in the food-finding test than like the ob/ob or ob/ob+carrier cohort. Potential local sites of leptin production and action were identified with immunohistochemistry and in situ hybridization in epithelial and gland cells of the olfactory and nasal mucosae. Our results strongly suggest that leptin acting through leptin receptors modulates olfactory-mediated pre-ingestive behavior.     

Milk intakes of genetically obese (ob/ob) and lean mouse pups differ with enhanced milk supply

Although obese (C57Bl/6J, ob/ob) pups have greater avidity for nonnutritive suckling than leans as early as 15 days postpartum, previous research has not found differences in milk intake between ob/ob and lean mice during the preweaning period. Because ob/ob pups suckle longer than leans, their perseveration should enhance their opportunity to ingest milk if (a) maternal milk supply is not limited and (b) longer sucking durations reflect increased pup willingness to ingest milk. Accordingly, the present study was designed to evaluate the milk intake of ob/ob and lean pups when they had access to an enhanced supply of maternal milk. Intact litters of pups, from heterozygous lean (ob/+) parents, were randomly assigned to be tested at either 6, 12, or 18 days. Pups were neither dam- nor milk-deprived before being cross-fostered successively to milk-replete surrogate dams for 60 min each. Obese pups showed a greater percentage body weight gain (the index of milk intake) than leans did, with younger pups showing larger increments than 18-day-olds. Although early adiposity in ob/ob pups may not rely on increased intake in the single-dam, nest situation, these data emphasize an early predisposition to overeating in this mutant.