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.     

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.     

Leptin improves pulmonary bacterial clearance and survival in ob/ob mice during pneumococcal pneumonia

The adipocyte-derived hormone leptin is an important regulator of appetite and energy expenditure and is now appreciated for its ability to control innate and adaptive immune responses. We have reported previously that the leptin-deficient ob/ob mouse exhibited increased susceptibility to the Gram-negative bacterium Klebsiella pneumoniae. In this report we assessed the impact of chronic leptin deficiency, using ob/ob mice, on pneumococcal pneumonia and examined whether restoring circulating leptin to physiological levels in vivo could improve host defences against this pathogen. We observed that ob/ob mice, compared with wild-type (WT) animals, exhibited enhanced lethality and reduced pulmonary bacterial clearance following Streptococcus pneumoniae challenge. These impairments in host defence in ob/ob mice were associated with elevated levels of lung tumour necrosis factor (TNF)-alpha, macrophage inflammatory peptide (MIP)-2 [correction added after online publication 28 September 2007: definition of MIP corrected], prostaglandin E(2) (PGE(2)), lung neutrophil polymorphonuclear leukocyte (PMN) counts, defective alveolar macrophage (AM) phagocytosis and PMN killing of S. pneumoniae in vitro. Exogenous leptin administration to ob/ob mice in vivo improved survival and greatly improved pulmonary bacterial clearance, reduced bacteraemia, reconstituted AM phagocytosis and PMN H(2)O(2) production and killing of S. pneumoniae in vitro. Our results demonstrate, for the first time, that leptin improves pulmonary bacterial clearance and survival in ob/ob mice during pneumococcal pneumonia. Further investigations are warranted to determine whether there is a potential therapeutic role for this adipokine in immunocompromised patients.     

Autonomic dysregulation in ob/ob mice is improved by inhibition of angiotensin-converting enzyme

The leptin-deficient ob/ob mice are insulin resistant and obese. However, the control of blood pressure in this model is not well defined. The goal of this study was to evaluate the role of leptin and of the renin-angiotensin system in the cardiovascular abnormalities observed in obesity using a model lacking leptin. To this purpose, we measured blood pressure in ob/ob and control animals by radiotelemetry combined with fast Fourier transformation before and after both leptin and enalapril treatment. Autonomic function was assessed pharmacologically. Blood pressure during daytime was slightly higher in the ob/ob compared to control mice, while no difference in heart rate was observed. Blood pressure response to trimetaphane and heart rate response to metoprolol were greater in ob/ob mice than in control littermates indicating an activated sympathetic nervous system. Heart rate response to atropine was attenuated. Baroreflex sensitivity and heart rate variability were blunted in ob/ob mice, while low frequency of systolic blood pressure variability was found increased. Chronic leptin replacement reduced blood pressure and reversed the impaired autonomic function observed in ob/ob mice. Inhibition of angiotensin-converting enzyme by enalapril treatment had similar effects, prior to the loss of weight. These findings suggest that the renin-angiotensin system is involved in the autonomic dysfunction caused by the lack of leptin in ob/ob mice and support a role of this interplay in the pathogenesis of obesity, hypertension, and metabolic syndrome.     

Pulmonary Mycobacterium tuberculosis infection in leptin-deficient ob/ob mice

The development of active tuberculosis after infection with Mycobacterium tuberculosis is almost invariably caused by a persistent or transient state of relative immunodeficiency. Leptin, the product of the obese (ob) gene, is a pleiotropic protein produced mainly by adipocytes and is down-regulated during malnutrition and starvation, conditions closely connected with active tuberculosis. To investigate the role of leptin in tuberculosis, we intranasally infected wild-type (Wt) and leptin-deficient ob/ob mice with live virulent M. tuberculosis. Ob/ob mice displayed higher mycobacterial loads in the lungs after 5 and 10 weeks of infection, although the difference with Wt mice remained 1 log of M. tuberculosis colony forming unit. Nevertheless, ob/ob mice were less able to form well-shaped granuloma and lung lymphocyte numbers were reduced compared with Wt mice early during infection. In addition, ob/ob mice had a reduced capacity to produce the protective cytokine IFNgamma at the site of the infection early during infection and upon antigen-specific recall stimulation, and showed reduced delayed-type hypersensitivity reaction to intra-dermal tuberculin purified protein derivative. Leptin replacement restored the reduced IFNgamma response observed in ob/ob mice. Mortality did not differ between ob/ob and Wt mice. These data suggest that leptin plays a role in the early immune response to pulmonary tuberculosis.     

Upregulated Expression of AQP 7 in the Skeletal Muscles of Obese ob/ob Mice

Aquaporin (AQP) is suggested to be regulated by leptin through the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway. AQP7 and AQP9 are membrane proteins with water and glycerol channels, the latter of which is essential for triglyceride synthesis. We conjectured that the expression of AQP7 and AQP9 would be altered in the skeletal myofibers in obese leptin deficient ob/ob mice as compared with that of wild mice. RNA and protein levels were studied in the quadriceps femoris muscles of ob/ob and wild mice. Real time quantitative RT-PCR analysis showed that mouse AQP7 mRNA levels in skeletal muscles were significantly higher in ob/ob mice than in wild mice (P<0.01), whereas mouse AQP9 mRNA level was not different between the two groups (P>0.05). Histologically the type 1 myofibers of ob/ob mice contained numerous lipid droplets in oil red O stain samples. Immunohistochemical staining of ob/ob mouse muscles revealed enhanced expression of AQP7 at myofiber surface membranes, while AQP9 expression appeared to be similar to that of wild mice. The findings suggest that the upregulated expression of AQP7 in ob/ob mouse muscles facilitates the secretion of glycerol from myocytes.     

Mice bearing the ob/ob mutation have impaired immunity.

The obese mutant mouse C57BL/6J ob/ob showed impaired ability to reject skin grafts or react to a contact-sensitising agent in comparison with littermate controls (either +/ob or +/+). Ability of spleen cells from mice bearing the ob/ob mutation to produce a graft-versus-host reaction in C57BL/6J X DBA/2J F1 hybrid mice was not impaired.     

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.     

Longitudinal evaluation of intramyocellular lipids (IMCLs) in tibialis anterior muscle of ob/ob and ob/+ control mice using a cryogenic surface coil at 9.4 T

Insulin resistance is a central feature of type II diabetes and is associated with alterations in skeletal muscle lipid metabolism, which manifest themselves, in part, in increased intramyocellular lipid (IMCL) accumulation. The objective of this study was to assess noninvasively the levels of IMCL longitudinally in the tibialis anterior muscle of Lep(ob) /Lep(ob) (ob/ob) mice, a genetic model of obesity and mild diabetes, and Lep(ob) /+ (ob/+) heterozygous control animals, using (1) H MRS at 9.4 T. The use of a cryogenic surface coil transceiver leads to significant increases in sensitivity. Method implementation included the assessment of the reproducibility and spatial heterogeneity of the IMCL signal and the determination of T(2) relaxation times, as IMCL levels were expressed relative to the total creatine signal, and therefore the signal ratios had to be corrected for differences in T(2) relaxation. IMCL levels were found to be significantly higher in ob/ob mice relative to ob/+ heterozygous control mice that do not develop disease. An increase in IMCL levels was observed for ob/ob mice until weeks 16/17; after this time point, IMCL levels decreased again, reaching final levels that were slightly higher than the initial values. These noninvasively detected alterations in skeletal muscle lipid metabolism in ob/ob mice were accompanied by a transient increase in plasma insulin concentrations. This study indicates that IMCL may be reliably assessed in mouse tibialis anterior muscle using a cryogenic surface coil, implying that (1) H MRS at 9.4 T represents a useful technology for the noninvasive measurement of changes in lipid metabolism in the skeletal muscle that accompany obesity.     

Reduced cholesterol accumulation by leptin deficient (ob/ob) mouse macrophages

Objective Although presenting many aspects of the metabolic syndrome, leptin deficient (ob/ob) mice do not spontaneously develop atherosclerosis. To examine the role of leptin in foam cell formation we analyzed ob/ob leukocyte inflammation markers and macrophage cholesterol accumulation. Methods Resident and thioglycollate (TG) elicited peritoneal cells of ob/ob and wildtype mice were studied. Activation markers, scavenger receptors (SR) and cholesterol accumulation were analyzed using flow cytometry and Taqman analysis. Cytokines, haptoglobin, adiponectin and amyloid A levels were analyzed with ELISA. Results Macrophages of ob/ob mice had reduced expression of MHC class II, CD11b, CD40, SR-A and CD36 and reduced cholesterol accumulation in vitro. Plasma haptoglobin was increased and T-cell IFNγ was reduced in ob/ob mice. Peritoneal TG instillation induced an unexpectedly weak inflammatory response in ob/ob mice. Conclusions The ob/ob mice had a reduced inflammatory response and reduced macrophage cholesterol accumulation in vitro. The data suggest decreased foam cell formation and atherosclerosis development in ob/ob mice. Received 27 January 2006; returned for revision 27 February 2006; accepted by A. Falus 27 March 2006     

A morphological and immunohistochemical investigation of endocrine pancreata from obese ob+/ob+ mice.

There is a marked difference in insulin secretion between the ob+/ob+ obese mouse and its non-obese littermate. Numerous peptides have been implicated in the modification of postprandial insulin secretion. In this study, the morphological and immunohistochemical studies of the genetically obese mouse (ob+/ob+) pancreata were compared with control littermates. Additionally, the distribution of gastric inhibitory polypeptide, somatostatin, glucagon, and insulin immunoreactive cells was also quantitated. Hyperglycemia and hyperinsulinemia were verified in the obese mice. The control animals had some islets and ductules with mononuclear infiltrations of a possible immune character. The obese individuals had a marked increase in both number and size of the islets of Langerhans compared with lean controls. The insulin immunocytochemical reaction in the obese pancreatic beta-cells was weaker than that of controls, as was the aldehyde-fuchsin reaction. The glucagon, gastric inhibitory polypeptide, and somatostatin containing cells were intermingled with the beta-cells. In contrast, the control animals showed a peripheral localization of these cell types. The morphometric analysis of the obese pancreas showed a decreased proportion of non-beta cells within the islets but not in total pancreatic volume in comparison with controls. The obese mouse also had cavities filled with eosin-stained material among numerous beta-cells. No complete epithelial lining distinguished these formations from the surrounding islet cells. The content of the cavities was not stained by any of the immunocytochemical reactions applied. In conclusion, the pancreatic islets of the ob+/ob+ mouse show marked differences in both morphological and immunocytochemical characteristics if compared with control littermates. These differences in architecture may be related to the eventual development of diabetes mellitus in the ob+/ob+ mouse.     

Regulation of myoplasmic Ca(2+) in genetically obese (ob/ob) mouse single skeletal muscle fibres

The present study examined whether calcium handling in skeletal muscle fibres from ob/ob mice was abnormal compared to normal mice. Simultaneous measurements of free myoplasmic calcium and force were made in mouse single intact muscle fibres at rest, during repetitive stimulation and for 30 min afterwards. Fibres were subjected to two bouts of intermittent tetanic contractions 1 h apart. The first bout consisted of 50 tetani only, while during the second bout stimulation was continued until force fell to 40% of control. During a bout of 50 repeated contractions, muscle fibres from ob/ob mice were unable to maintain basal calcium and tetanic calcium transients. During a second series of contractions, muscle fibres from ob/ob mice showed a marked improvement in calcium handling compared to the first series but still fatigued more rapidly than control fibres. It is concluded that calcium handling in skeletal muscle fibres from ob/ob mice is abnormal compared to fibres from normal mice and this contributes to premature fatigue.     

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.     

去铁敏阻抑ob/ob小鼠海马脑区的Tau蛋白过度磷酸化

目的研究去铁敏(DFO)对ob/ob小鼠脑内Tau过度磷酸化的影响,以探讨铁沉积是否涉及糖尿病(DM)的神经病理。方法繁育12只6月龄ob/ob小鼠,随机分为DFO组和对照组(n=6),分别给予腹腔注射DFO(100 mg·kg^-1)或空白溶剂15d。采用免疫组织化学和Western blot方法,检测小鼠海马脑区的Tau蛋白磷酸化及其调控蛋白激酶和磷酸酶的表达变化;DAB增强的Perl’s染色检测海马铁离子的分布。结果DFO处理后,ob/ob小鼠海马脑区Tau蛋白无明显变化,而在Ser396和Thr231位点的磷酸化水平降低。相应地,DFO组小鼠脑内蛋白激酶GSK3β和CDK5的活性显著下调,磷酸酶PP2A及其活性上调。另外,DFO组小鼠海马脑区的铁染色强度减弱。结论DFO能够有效改善ob/ob小鼠海马脑区的Tau病理,为揭示铁参与DM诱发的神经病理提供了新证据。     

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.     

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.     

硫化氢在ob/ob小鼠皮肤创面愈合中的作用与调控机制

目的:观察硫化氢(H2S)对ob/ob小鼠皮肤创面愈合的影响并探讨其作用机制。方法:将ob/ob小鼠随机分为生理盐水组、胰岛素组和NaHS(H2S供体)组,C57BL/6小鼠作为对照组,构建小鼠背部皮肤创面模型。干预后检测各组H2S释放量;用Western blot检测胱硫醚γ-裂解酶(CSE)及基质金属蛋白酶-9(MMP-9)蛋白的表达差异;用RT-qPCR检测CSE的mRNA表达变化;使用免疫组织化学法检测中性粒细胞及单核/巨噬细胞的浸润数量;使用ELISA检测肿瘤坏死因子(TNF)-α和白细胞介素(IL)-6的水平;用Masson染色检测胶原沉积情况。结果:ob/ob小鼠皮肤创面肉芽组织中H2S释放及CSE蛋白、mRNA的表达水平以及胶原沉积显著低于C57BL/6小鼠(P0.05)。外源性H2S可加速ob/ob小鼠皮肤创面愈合(P0.05),增加胶原沉积。ob/ob小鼠创面中性粒细胞及单核/巨噬细胞浸润数量,TNF-α、IL-6的水平及MMP-9蛋白表达水平显著增加(P0.05),NaHS组显著降低。结论:H2S可显著改善糖尿病难愈性溃疡的愈合,作用机制可能与其抗炎作用有关。     

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.     

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.     

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.