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.     

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.     

Altered myoplasmic Ca2+ handling in rat fast-twitch skeletal muscle fibres during disuse atrophy

Calcium-dependent signalling pathways are believed to play an important role in skeletal muscle atrophy, but whether intracellular Ca²⁺ homeostasis is affected in that situation remains obscure. We show here that there is a 20% atrophy of the fast-type flexor digitorum brevis (FDB) muscle in rats hind limb unloaded (HU) for 2 weeks, with no change in fibre type distribution. In voltage-clamp experiments, the amplitude of the slow Ca²⁺ current was found similar in fibres from control and HU animals. In fibres loaded with the Ca²⁺ dye indo-1, the value for the rate of [Ca²⁺] decay after the end of 5–100-ms-long voltage-clamp depolarisations from ?80 to +10 mV was found to be 30–50% lower in fibres from HU animals. This effect was consistent with a reduced contribution of both saturable and non-saturable components of myoplasmic Ca²⁺ removal. However, there was no change in the relative amount of parvalbumin, and type 1 sarco-endoplasmic reticulum Ca²⁺-ATPase was increased by a factor of three in the atrophied muscles. Confocal imaging of mitochondrial membrane potential showed that atrophied FDB fibres had significantly depolarized mitochondria as compared to control fibres. Depolarization of mitochondria in control fibres with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone induced a slowing of the decay of [Ca²⁺] transients accompanied by an increase in resting [Ca²⁺] and a reduction of the peak amplitude of the transients. Overall results provide the first functional evidence for severely altered intracellular Ca²⁺ removal capabilities in atrophied fast-type muscle fibres and highlight the possible contribution of reduced mitochondrial polarisation.     

Regional blood flow in genetically obese (ob/ob) mice

1. Experiments have been undertaken on 11 decerebrate cats to investigate the effects of natural vestibular stimulation on the activity of cerebellar fastigial neurons. 2. From recordings in the rostral portion of the nucleus during sinusoidal lateral (roll) and horizontal (yaw) rotation, distinctive patterns of response were observed. 3. The majority of neurons sensitive to vestibular stimulation showed responses to a single modality of vestibular activation. During lateral tilt some neurones showed positional sensititivy, others gave responses related tothe velocity of movement. Other neurones responded in phase with the velocity of movement in the horizontal plane. 4. Aside from these neuronal responses, others provided indications of a convergence of inputs from different sets of vestibular receptors. In particular, several neurons showed a pattern of response that indicated tht they received inputs from otolith receptors and ampullar receptors of the vertical canal. At low velocities of movement their response was positional but with inreasing velocity the magnitude of the response increased and there was a marked phase shift of the discharge towards head velocity. 5. Neurons responding to horizontal rotation often showed positional responses during lateral tilt. There were also indications of a convergence of ampullar inputs from both vertical and horizontal canals. 6. The neural pathways mediating these resonses are discussed in consideration of previous neuroanatomical and neurophysiological data. We consider it likely that several pathways may act to evoke the patterns of response observed, and a role of the cerebellar cortex is indicated.     

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.     

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.     

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.     

Activation of Ca(2+)-dependent protein kinase II during repeated contractions in single muscle fibres from mouse is dependent on the frequency of sarcoplasmic reticulum Ca(2+) release

Aim: To investigate the importance and contribution of calmodulin‐dependent protein kinase II (CaMKII) activity on sarcoplasmic reticulum (SR) Ca²⁺‐release in response to different work intensities in single, intact muscle fibres. Methods: CaMKII activity was blocked in single muscle fibres using either the inhibitory peptide AC3‐I or the pharmacological inhibitor KN‐93. The effect on tetanic force production and [Ca²⁺]_i was determined during work of different intensities. The activity of CaMKII was assessed by mathematical modelling. Results: Using a standard protocol to induce fatigue (50× 70 Hz, 350 ms duration, every 2 s) the number of stimuli needed to induce fatigue was decreased from 47 ± 3 contractions in control to 33 ± 3 with AC3‐I. KN‐93 was a more potent inhibitor, decreasing the number of contractions needed to induce fatigue to 15 ± 3. Tetanic [Ca²⁺]_i was 100 ± 11%, 97 ± 11% and 67 ± 11% at the end of stimulation in control, AC3‐I and KN‐93 respectively. A similar inhibition was obtained using a high intensity protocol (20× 70 Hz, 200 ms duration, every 300 ms). However, using a long interval protocol (25× 70 Hz, 350 ms duration, every 5 s) no change was observed in either tetanic [Ca²⁺]_i or force when inhibiting CaMKII. A mathematical model used to investigate the activation pattern of CaMKII suggests that there is a threshold of active CaMKII that has to be surpassed in order for CaMKII to affect SR Ca²⁺ release. Conclusion: Our results show that CaMKII is crucial for maintaining proper SR Ca²⁺ release and that this is regulated in a work intensity manner.     

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.     

Intracellular ATP measured with luciferin/luciferase in isolated single mouse skeletal muscle fibres

The firefly luciferin/luciferase reaction was utilized to monitor intracellular ATP concentration ([ATP](i)). Single fibres of mouse skeletal muscle were dissected and injected with luciferase. Luciferin was added to the perfusate and light emission from the fibres was monitored as an indication of [ATP](i). Inhibition of oxidative phosphorylation with cyanide and anaerobic glycolysis with iodoacetate caused light emission to fall to zero within 10 min and the fibres developed a rigor contraction. Inhibition of creatine kinase with 2,4-dinitro-1-fluorobenzene produced a small transient fall in light emission in association with each tetanus. Muscle fibres were fatigued by repeated tetani and 5/12 fibres showed a fall in light emission in the late phase of fatigue. If fibres were allowed to recover from fatigue in the absence of glucose and then restimulated in the absence of glucose they fatigued much more rapidly. However, such fibres showed no obvious change in light emission. We conclude that the luciferin/luciferase system can be used to monitor [ATP](i) in functioning single skeletal muscle cells. A depletion of global [ATP](i) is not observed in all fatiguing fibres and cannot be the sole cause of the final phase of fatigue.     

Ca2+-activated force-generating properties of mammalian skeletal muscle fibres: histochemically identified single peeled rabbit fibres

Summary Single peeled (sarcolemma removed) rabbit skeletal muscle fibres, identified histochemically from their myofibrillar ATPase and oxidative staining pattterns, were characterized according to their Ca²⁺-activated steady-state force-generating properties at normal intracellular pH (7.0) and under acidotic (pH 6.5) conditions. Maximum force-generating capacity of each fibre was assessed by measuring steady-state isometric force generation at saturating Ca²⁺ concentration at both pH values. The Ca²⁺ sensitivity of each fibre was ascertained by determining the percentage of maximum force generated at each of several subsaturating Ca²⁺ concentrations at both pH values. Fibres were selected from soleus, tibialis anterior and adductor magnus muscles. At subsaturating Ca²⁺ concentrations only two functional groups of fibres were distinguishable, corresponding to the histochemical classifications type I and type II. Type I fibres were more sensitive to Ca²⁺ and less depressed by acidosis than type II fibres in the subsaturating range of Ca²⁺ concentrations. At saturating Ca²⁺ concentrations, the acidotic depression of maximum force was significantly less for type I fibres than type II nonoxidative fibres regardless of their muscle of origin. Type II oxidative fibre maximum force properties depended upon the muscle of origin and demonstrated subgroups of these fibres that were different from type II nonoxidative fibres and similar to type I fibres.     

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.     

The kappa opioid receptor, ingestive behaviors and the obese mouse (ob/ob)

Recent studies have suggested a role for the kappa opiate receptor and its endogenous ligand, dynorphin, in the central regulation of appetite. In this study we found that the ob/ob mouse was mildly resistant to the ability of three kappa agonists, viz, butorphanol, tifluadom, and ketocyclazocine to induce food intake. In addition, we could find no change in ir-dynorphin levels in 7 areas for the central nervous system. These findings do not provide evidence for a role of kappa opioid feeding system in the pathogenesis of obesity in the ob/ob mouse.     

Hunger and satiety in genetically obese mice (C57BL/6J-ob/ob)

To determine the mechanism for hyperphagia in genetically obese mice (C57BL/6J-ob/ob), several experiments were conducted on the ability of these mice to respond to caloric deficits and surpluses. Presentation of food or sugar reduces subsequent operant licking in both obese and lean mice. When given sugar solutions, evaporated milk, or sweetened non-fat milk, both obese and lean mice reduce food intake to compensate for the calories obtained from the solutions. These findings indicate that genetically obese mice respond normally to caloric surpluses. Obese mice respond to food deprivation (caloric deficit) by increasing subsequent food intake but they do so more slowly than controls.     

Changes in mitochondrial Ca2+ detected with Rhod-2 in single frog and mouse skeletal muscle fibres during and after repeated tetanic contractions

The present study investigated mitochondrial Ca²⁺ uptake and release in intact living skeletal muscle fibres subjected to bouts of repetitive activity. Confocal microscopy was used in conjunction with the Ca²⁺-sensitive dye Rhod-2 to monitor changes in mitochondrial Ca²⁺ in single Xenopus or mouse muscle fibres. A marked increase in the mitochondrial Ca²⁺ occurred in Xenopus fibres after 10 tetani applied at 4 s intervals. The mitochondrial Ca²⁺ continued to increase with increasing number of tetani. After the end of tetanic stimulation, mitochondrial Ca²⁺ declined to 50% of the maximal increase within 10 min and thereafter took up to 60 min to return to its original value. Depolarization of the mitochondria with FCCP greatly attenuated the rise in the mitochondrial Ca²⁺ evoked by repetitive tetanic stimulation. In addition, FCCP slowed the rate of decay of the tetanic Ca²⁺ transient which in turn led to an elevation of resting cytosolic Ca²⁺. Accumulation of Ca²⁺ in the mitochondria was accompanied by a modest mitochondrial depolarization. In contrast to the situation in Xenopus fibres, mitochondria in mouse toe muscle fibres did not show any change in the mitochondrial Ca²⁺ during repetitive stimulation and FCCP had no effect on the rate of decay of the tetanic Ca²⁺ transient. It is concluded that in Xenopus fibres, mitochondria play a role in the regulation of cytosolic Ca²⁺ and contribute to the relaxation of tetanic Ca²⁺ transients. In contrast to their important role in Xenopus fibres, mitochondria in mouse fast-twitch skeletal fibres play little role in Ca²⁺ homeostasis.     

Early effects of denervation on Ca(2+)-handling proteins in skeletal muscle

The adaptive response of skeletal muscle fibres depends on a variety of biological factors including loading conditions and neuromuscular activity. An extreme type of atrophy-inducing change in contractile activity is represented by the physical disconnection between the motor nerve and its respective fibre unit. Since fibre type alterations have a striking effect on the Ca(2+)-regulatory apparatus, we have investigated the fate of a key Ca(2+)-pump and essential Ca(2+)-binding proteins in extensor digitorum longus specimens two weeks after nerve crush or complete denervation. In contrast to increased levels of sarcalumenin, immunoblotting revealed that the expression of the fast SERCA1 Ca(2+)-ATPase isoform is drastically decreased and fast calsequestrin is slightly reduced. Analysis of myosin heavy chain isoforms agreed with this result and showed a fast-to-slow fibre type shifting process following denervation. Hence, changes in muscle activity appear to have a profound effect on the abundance and isoform expression pattern of Ca(2+)-handling elements.     

Decrease in particle-induced osteolysis in obese (ob/ob) mice

There may be variability in the susceptibility of different individuals to osteolysis from wear debris, and it is not clear whether some individuals may have a genetic predisposition for a more marked osteolytic response. The purpose of this study in mice was to determine whether genetically determined obesity can alter the response to particulate debris. Polyethylene particles were implanted onto the calvaria of seven wild-type mice and seven obese mice (ob/ob). Calvaria from unimplanted wild-type and obese mice served as controls. Calvaria were harvested after 7 days, stained with toluidine blue and for tartrate-specific alkaline phosphatase, and analyzed by histomorphometry. The osteoclast number per mm total bone perimeter was 8.000+/-3.464 in wild-type animals with particles and 2.857+/-1.676 in ob/ob animals with particles (p=0.002; Fisher's PLSD). Bone resorption was 1.895+/-0.713 mm/mm(2) in wild-type animals with particles and 1.265+/-0.494 mm/mm(2) in ob/ob animals with particles (p=0.0438; Fisher's PLSD). Particles induced a diminished osteolytic response in genetically determined obese mice, suggesting that obesity may have a protective role against particle-induced bone resorption-similar to obesity and osteoporosis. These important new findings may help to stimulate clinical studies which may define criteria to better identify patients at risk to develop particle-induced osteolysis.