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.     

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.     

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.     

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.     

Thermal preference behavior of genetically obese (ob/ob) and genetically lean (+/?) mice

Chronically lower colonic temperatures (T_cs) of genetically-obese (ob/ob) mice at ambient temperatures below thermoneutrality have led to speculation that these mutants regulate a lowered thermal setpoint relative to lean mice. Previous experiments, however, have not provided an opportunity for obese mice to exhibit compensatory thermoregulatory behaviors which might reinstate normal body temperature. In the present experiment, adult obese and lean (+/?) mice were tested at room temperature (25°C) on a copper bar, thermal gradient to determine what temperatures they would select and what effect their selection would have on their T_cs. The data revealed that ob/obs were more frequently observed within 25–35°C locales than were lean controls, which spent more time in the below-25°C zone (p<0.005). Ob/obs also raised their T_cs to pretest values of leans' T_cs, although ob/obs' T_cs remained significantly lower than those of leans at the conclusion of testing. These data suggest that the hypothermia exhibited by the ob/ob may reflect both the absence of the opportunity to behaviorally thermoregulate and a genetic defect in thermogenesis.     

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.     

Urocortin reduces oxygen consumption in lean and ob/ob mice

A vast number of intensive studies have been undertaken to clarify the mechanisms of energy balance. This study was undertaken to investigate the effect of urocortin, an endogenous ligand for corticotropin-releasing factor (CRF) type 2 receptor, on oxygen consumption in lean and genetically obese (ob/ob) mice. Oxygen consumption was measured after intraperitoneal injection in unrestrained mice at an environmental temperature of 22 degrees C of one of the following: urocortin, deamidated form of urocortin (urocortin OH) or CRF. The intraperitoneal injection of urocortin (0.3-3 nmol) dose-dependently decreased oxygen consumption in lean mice. The inhibitory effect induced by urocortin was more potent than that induced by CRF or urocortin OH. The ranking potency was urocortin > urocortin OH > CRF. Urocortin significantly reduced oxygen consumption in ob/ob mice as well as in lean mice. These results suggest that urocortin decreases oxygen consumption, and that the CRF type 2 receptor may influence energy balance in lean and ob/ob mice.     

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.     

Role of sympathetic nervous system in immobilization- and cold-induced brown adipose tissue thermogenesis in rats

The role of the sympathetic nervous system in 10-min cold (5 degrees C)- or 2-min immobilization-induced thermogenesis in brown adipose tissue (BAT) was studied in warm (25 degrees C)-acclimated rats. Both cold- and immobilization-stresses increased heat production (M), interscapular brown adipose tissue temperature ( Tbat ), and colonic temperature ( Tcol ). Resulting from both stresses, the increase in Tbat was greater than that in Tcol , the differences (delta Tbat ) becoming approximately 0.48 and 0.46 degrees C by the cold exposure and the immobilization, respectively. After sympathectomy, Tbat and delta Tbat did not change on immobilization but increased significantly on the cold exposure. Delta Tbat was 0.31 degrees C in the sympathectomized rats at the end of the cold exposure period. Immobilization-induced BAT thermogenesis may be mainly controlled by the sympathetic nervous system. On the other hand cold-induced BAT thermogenesis seems to be controlled by certain hormonal factors as well as the sympathetic nervous system.     

Short photoperiod stimulates brown adipose tissue growth and thermogenesis but not norepinephrine turnover in Syrian hamsters

This experiment examined the effect of short photoperiod on food intake, body weight, carcass composition, and brown adipose tissue (BAT) activity in female Syrian hamsters (Mesocricetus auratus). BAT function was assessed by measuring sympathetic nervous system (SNS) activity in BAT (estimated by the rate of norepinephrine (NE) turnover), BAT mitochondrial content (estimated by cytochrome c oxidase activity), and BAT mitochondrial proton conductance (estimated by guanosine-5'-diphosphate (GDP) binding to isolated BAT mitochondria). Food intake and body weight were both increased in short photoperiod-housed hamsters (8-hr light, 16-hr dark; LD 8:16) when compared to those of the long photoperiod-housed controls (LD 16:8). The weight gain was entirely due to an increase in carcass lipid. Interscapular BAT (IBAT) pads from short photoperiod-housed hamsters were 53% heavier and contained comparably more protein and DNA. Short photoperiod produced a 118% increase in BAT cytochrome c oxidase activity and a 41% increase in specific mitochondrial GDP binding. Whether expressed per mg wet tissue or per pad, neither the endogenous concentration of NE nor its rate of turnover were changed by short photoperiod exposure. These results demonstrate a dissociation of BAT thermogenesis from SNS activity in BAT from short photoperiod-housed Syrian hamsters.     

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.     

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.     

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.     

Effects of the obese (ob/ob) genotype on spleen cell immune function.

Spleen cells from mice homozygous for the obese (ob) mutation killed DBA/2 mastocytoma target cells less well than spleen cells from lean littermates or unrelated age-and sex-matched controls of the same strain. Killing was impaired only when the attacker cells were primed in vivo, not following in vitro priming. Hence the effect of the ob/ob genotype is not to produce an irreversible functional change in the lymphocyte, but rather to produce an environment in which lymphocytes are less able to react to priming antigen. Not only were the spleen cells of in vivo primed obese mice less active than those of lean controls, but also their number per spleen was significantly decreased. Such a quantitive difference was no longer found in adrenalectomised animals, but the qualitative difference in spleen cell cytotoxic activity still occurred. This suggests that adrenocortical hyperfunction may affect immune function in obese mice, without necessarily being the only factor in the in vivo environment of obese mouse spleen cells capable of depressing cellular immune reactivity.     

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.     

Effects of cold and immobilization stress on noradrenaline turnover in brown adipose tissue of rat

Noradrenaline (NA) turnover of the interscapular brown adipose tissue (BAT) was determined in order to evaluate a role of sympathetic NA of this tissue in an enhanced nonshivering thermogenesis which had been previously evidenced in the repetitively stressed rats by immobilization (daily 3-h immobilization for 4 weeks) and the cold-acclimated ones (5 degrees C, 4 weeks). The disappearance rate of NA from the BAT following blockade of NA synthesis with alpha-methyl-p-tyrosine was adopted for estimation of NA turnover of the tissue. Cold acclimation increased both fractional turnover rate (%/h) (k) and turnover rate (ng/(g BAT.h)). Repetitive immobilization stress also elevated turnover rate, but not k. In the warm non-stressed controls acute cold exposure to -5 degrees C and acute immobilization stress elevated the turnover rate. The effect of cold exposure was significantly greater than that of immobilization stress for both indices of NA turnover. In the cold-acclimated rats acute cold exposure increased k as well as turnover rate, but not acute immobilization stress. In the repetitively immobilized rats both acute cold exposure and acute immobilization stress elevated k and turnover rate. These results indicate that immobilization enhances sympathetic activity of thermogenic tissue, BAT. The results also suggest that the extent of sympathetic participation is not necessarily the same between the cold-acclimated and the stressed rats.     

Increased responsiveness of ventromedial hypothalamic neurons to norepinephrine in obese versus lean mice: relation to the metabolic syndrome

Studies of the effects of acute and chronic norepinephrine (NE) infusion into the ventromedial hypothalamus (VMH) of rodents indicate important roles for VMH NE activities in the development of the obese-glucose intolerant state. Moreover, elevated endogenous levels of NE and/or its metabolites have been observed in a variety of obese-glucose intolerant animal models. We therefore investigated the VMH neuronal electrophysiologic responsiveness to iontophoretically applied NE in lean-euglycemic and obese-hyperglycemic mice. Additionally, the effect of dopamine agonist treatment (which reduces obesity and hyperglycemia) on VMH responsiveness to NE was examined in obese-hyperglycemic mice. Obese (ob/ob) mice were treated daily for 14 days with either bromocriptine (BC, D2 agonist) (10 mg/kg) plus SKF38393 (SKF, D1 agonist) (20 mg/kg) or vehicle. Lean mice were also similarly treated with vehicle. Twenty-seven hours following the final treatment, mice were anesthetized to obtain electrophysiologic responses of glutamate activated VMH neurons to local NE administration. In all three study groups, NE administration inhibited glutamate evoked neuronal activity in the majority (90%) of recorded neurons. No response to NE was observed in the remaining 10% of neurons. Also within all three groups there existed two patterns of response to NE; a) long duration (>2 min) and low threshold (<20 nA) and b) short duration and high threshold. Relative to lean mice, obese mice exhibited a significant 70% increase in average duration of response, 3-fold increase in percent neurons with long duration of response, and 2-fold increase in percent neurons with low threshold of response. BC/SKF treatment of obese mice significantly reduced the percent VMH neurons with long duration and low threshold of response to NE to resemble the VMH neuronal responsiveness to NE observed in lean mice. Increased VMH responsiveness to NE is part of the endogenous neurophysiology of obese-hyperglycemic ob/ob mice. Taken together with previous findings mentioned above, the present results suggest that this increased VMH responsiveness to NE contributes to the pathophysiology of the obese-hyperglycemic state.     

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.     

Effect of acute cold-exposure on norepinephrine turnover and thermogenesis in brown adipose tissue and metabolic rate in MSG-induced obese mice

Mice treated neonatally with monosodium-L-glutamate (MSG) are known to develop into obese adults without hyperphagia, which are characterized by the reduced levels in the resting metabolic rate (RMR) and the thermogenesis of brown adipose tissue (BAT) in the thermoneutral environment. The present study revealed that an acute cold-exposure (5 degrees C, 1 h) of these animals resulted in the increase in norepinephrine turnover and mitochondrial-5'-diphosphate (GDP) binding in the interscapular BAT as well as the guanosine RMR, suggesting a normal thermogenic responsiveness of BAT to cold.