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.     

Classically conditioned hyperglycemia in the obese mouse

The obese (C57BL/6J ob/ob) mouse is a commonly used animal model of non-insulin-dependent diabetes mellitus. It has recently been demonstrated that this mouse is not consistently hyperglycemic, however, unless it is subjected to environmental stress. In the present study, hyperglycemia in obese mice was induced by classical conditioning. Obese diabetic mice and lean control animals were exposed to shaking stress. All animals developed hyperglycemia in response to shaking. To demonstrate classical conditioning, some obese and lean animals were exposed to a metronome prior to and during the shaking. Other animals were exposed to the metronome and shaking in a noncontingent fashion and one group of animals was exposed to the metronome without any exposure to shaking. All animals received seven exposures to one of the three above conditions over a 3-day period. On the 4th day all animals were exposed to the metronome alone, following which blood samples were drawn. Classical conditioning of stress hyperglycemia was demonstrated in obese, but not in lean, mice.     

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.     

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.     

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.     

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.     

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.     

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.     

饥饿、糖尿病和肥胖状态对小鼠肝脏SOCS2基因表达的影响

目的确定小鼠肝脏SOCS2基因在饥饿、糖尿病和肥胖状态下的表达水平,并初步研究SOCS2对糖异生的影响。方法动物分3组:C57BL/6J小鼠、对照组(饱食)和实验组(饥饿24 h);糖尿病模型小鼠db/db及对照小鼠db/m饱食;肥胖模型小鼠ob/ob及其对照C57BL/6J小鼠(饱食)。处死小鼠后提取肝脏RNA做反转录PCR,荧光实时定量PCR检测小鼠肝脏SOCS2及糖异生相关基因在3组小鼠中的表达水平;使用腺病毒表达系统在C57BL/6J小鼠原代肝细胞中过表达SOCS2,Western blot检测SOCS2蛋白的表达,葡萄糖生成实验检测糖输出。结果饥饿使C57BL/6J小鼠肝脏中SOCS2 mRNA水平下调,db/db和ob/ob小鼠肝脏SOCS2基因表达比其对照小鼠均明显下降(P0.05),调节糖异生的关键基因PGC-1α、PEPCK和G6Pase的mRNA水平均上升。在C57BL/6J小鼠原代肝细胞中过表达SOCS2,得到大小为Mr 23 000的蛋白,糖输出受到明显抑制。结论初步认定SOCS2可抑制C57BL/6J小鼠原代肝细胞糖异生,可能是治疗糖尿病的一个新靶点。     

Naltrexone potentiates glycemic responses during stress and epinephrine challenge in genetically obese mice

The genetically obese mouse (C57BL/6J ob/ob) is a commonly used animal model of non-insulin-dependent diabetes mellitus. These mice show exaggerated glycemic responses during behavioral stress and adrenergic stimulation, but the precise glucoregulatory mechanisms are not well characterized. The ob/ob mice have multiple endocrine abnormalities, including elevated pituitary and circulating beta-endorphin levels; and a relationship between hyperglycemia and altered opioid function has been suspected. We now report that opiate antagonism with naltrexone potentiates hyperglycemic responses during stress and epinephrine challenge in obese mice. This effect of opioid blockade suggests that endogenous opioids inhibit stress- and epinephrine-induced hyperglycemia in the genetically obese mouse.     

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.     

Insulin secretion by isolated perfused rat and mouse pancreas.

A method for isolation and perfusion of a pancreas preparation consisting of pancreas, stomach, proximal duodenum, and spleen is described. Basic characteristics of regulation of insulin secretion from the perfused pancreas isolated from rats, albino mice, obese mice (ob/ob), and black mice were identical. Viability and stability of the pancreas preparation during perfusion were maintained as documented by measurements of oxygen consumption of the pancreas preparation, perfusion pressure, and pH of the perfusion medium. The insulin-secretory capacity of the pancreas of different animal species was compared. Insulin secretion by the perfused rat and obese (ob/ob) mouse pancreas was much more potent than that by the pancreas of lean albino and lean black mice. D-Glucose-induced insulin secretion from the pancreas was decreased after fasting of the animals and was dependent on glucose concentration and presence of calcium in the perfusion medium. D-Glyceraldehyde, tolbutamide, D-mannose, dihydroxyacetone, L-leucine, and L-arginine also induced insulin secretion from the pancreas. D-Fructose, D-galactose, L-glucose, 3-O-methyl-D-glucose, N-acetylglucosamine, D-xylose, D,L-glyceric acid, pyruvate, L-lactate, and theophylline did not provoke insulin secretion.     

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.     

Ob/ob serum promotes a mesenchymal cell phenotype in B16BL6 melanoma cells

In 2009, malignant melanoma was responsible for approximately 9,000 deaths in the US. These deaths are often associated with aggressive metastasis to secondary sites such as the lungs. Epidemiological and animal studies suggest that obesity is a risk factor for melanoma. Others have shown that B16BL6 melanoma cells metastasize more aggressively in obese ob/ob than in lean mice. However, the mechanism by which obesity promotes B16BL6 melanoma metastasis in ob/ob mice has not been identified. In the present study, we used serum obtained from control and ob/ob leptin-deficient obese mice to determine if obese serum increases the aggressive phenotype of melanoma cells. Results showed that ob/ob serum has higher levels of resistin, insulin, tPAI1, IL-6, TNF-α, and MCP-1 compared to control serum. We showed that ob/ob serum increases the invasive ability of B16BL6 melanomas. To further determine the mechanism by which ob/ob serum increases the invasive ability of melanomas, we determined the effect of ob/ob and control serum on genes associated with the epithelial-to-mesenchymal transition (EMT). Cancer cells with a mesenchymal phenotype have a higher metastatic ability. Snai1 and Twist are genes that are strongly associated with EMT and metastasis of melanomas. Our results showed that ob/ob serum increases the expression of Snai1 and Twist. Moreover, ob/ob serum increased matrix metalloproteast 9 (MMP9) activity and decreased the expression of E-cadherin and the metastasis suppressor gene Kiss1. In summary, results suggest that obesity may increase the metastatic ability of melanoma by promoting a mesenchymal cell phenotype.     

Intermittent Hypoxia Increases Insulin Resistance in Genetically Obese Mice

Obstructive sleep apnoea, a syndrome that leads to recurrent intermittent hypoxia, is associated with insulin resistance in obese individuals, but the mechanisms underlying this association remain unknown. We utilized a mouse model to examine the effects of intermittent hypoxia on insulin resistance in lean C57BL/6J mice and leptin-deficient obese (C57BL/6J− Lep ^ob) mice. In lean mice, exposure to intermittent hypoxia for 5 days (short term) resulted in a decrease in fasting blood glucose levels (from 173 ± 11 mg dl⁻¹ on day 0 to 138 ± 10 mg dl⁻¹ on day 5, P < 0.01), improvement in glucose tolerance without a change in serum insulin levels and an increase in serum leptin levels in comparison with control (2.6 ± 0.3 vs. 1.7 ± 0.2 ng ml⁻¹, P < 0.05). Microarray mRNA analysis of adipose tissue revealed that leptin was the only upregulated gene affecting glucose uptake. In obese mice, short-term intermittent hypoxia led to a decrease in blood glucose levels accompanied by a 607 ± 136 % (   P < 0.01  ) increase in serum insulin levels. This increase in insulin secretion after 5 days of intermittent hypoxia was completely abolished by prior leptin infusion. Obese mice exposed to intermittent hypoxia for 12 weeks (long term) developed a time-dependent increase in fasting serum insulin levels (from 3.6 ± 1.1 ng ml⁻¹ at baseline to 9.8 ± 1.8 ng ml⁻¹ at week 12, P < 0.001) and worsening glucose tolerance, consistent with an increase in insulin resistance. We conclude that the increase in insulin resistance in response to intermittent hypoxia is dependent on the disruption of leptin pathways.     

Severely impaired insulin signaling in chronic wounds of diabetic ob/ob mice: a potential role of tumor necrosis factor-alpha

Wound-healing disorders are major complications of diabetes mellitus. Here, we investigated insulin-mediated signaling in nonwounded skin and in cutaneous tissue regeneration of healthy C57BL/6 and diabetes-impaired leptin-deficient obese/obese (ob/ob) mice. The insulin receptor (InsR) was abundantly expressed in wound margins and granulation tissue during acute healing in healthy mice. Remarkably, active signaling from the InsR, as assessed by phosphorylation of downstream targets such as protein tyrosine phosphatase-1B, glycogen synthase (GS), and GS kinase, was nearly absent in nonwounded and acutely healing skin from ob/ob mice. Systemic leptin administration to ob/ob mice reverted the diabetic phenotype and improved tissue regeneration as well as the impaired expression of InsR, insulin receptor substrate-1 and insulin receptor substrate-2, and downstream signaling (phosphorylation of GS kinase and GS) in late wounds and nonwounded skin of ob/ob mice. Importantly, tumor necrosis factor (TNF)-alpha was a mediator of insulin resistance in keratinocytes in vitro and in ob/ob wound tissue in vivo. Systemic administration of a monoclonal anti-TNF-alpha antibody (V1q) in wounded ob/ob mice attenuated wound inflammation, improved re-epithelialization, and restored InsR expression and signaling in wound tissue of ob/ob mice. These data suggest that InsR signaling in diabetes-impaired wounds is sensitive to inflammatory conditions and that anti-inflammatory approaches, such as anti-TNF-alpha strategies, improve diabetic wound healing.     

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.     

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.     

Functional abnormalities of islets of Langerhans of obese hyperglycemic mouse.

Glucose-induced insulin release was studied in vitro with isolated islets of Langerhans obtained from obese hyperglycemic C57Bl/6J-ob/ob (ob/ob) and lean C57Bl/6J-+/+ (control) mice. The threshold concentrations of glucose for insulin release were determined. In addition, the effect of total fast and of chronic food restriction on in vitro insulin release were studied. The following was observed: 1) with fasting, islet volume decreased. Islets obtained from ob/ob mice were larger than control islets, except for the chronic food restricted group. 2) Ob/ob islets were more sensitive to glucose than were controls in that the threshold for glucose-induced insulin release occured at lower glucose concentrations. 3) Fasting for 48 h completely abolished glucose-induced insulin release in control islets, whereas glucose-induced insulin release was maintained in 48-h and 7-day fasted ob/ob islets. 4) The increased glucose sensitivity of the ob/ob islets was maintained despite chronic food restriction.     

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.