Extracellular superoxide dismutase in tissues from obese (ob/ob) mice

We have examined the protein content and gene expression of three superoxide dismutase (SOD) isoenzymes in eight tissues from obese ob/ob mice, particularly placing the focus on extracellular-SOD (EC-SOD) in the white adipose tissue (WAT). Obesity significantly increased EC-SOD level in liver, kidney, testis, gastrocnemius muscle, WAT, brown adipose tissue (BAT), and plasma, but significantly decreased the isoenzyme level in lung. Tumor necrosis factor-α and interleukin-1β contents in WAT were significantly higher in obese mice than in lean control mice. Immunohistochemically, both WAT and BAT from obese mice could be stained deeply with anti-mouse EC-SOD antibody compared with those from lean mice. Each primary culture per se almost time-dependently enhanced EC-SOD production, and overtly expressed its mRNA. The loss of heparin-binding affinity of EC-SOD type C with high affinity for heparin occurred in kidney of obese mice. These results suggest that the physiological importance of this SOD isoenzyme in WAT may be a compensatory adaptation to oxidative stress.     

Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species.

The contents of extracellular superoxide dismutase, CuZn superoxide dismutase and Mn superoxide dismutase were determined in tissues from nine mammalian species. The pattern of CuZn superoxide dismutase distribution was similar in all species, with high activity in metabolically active organs such as liver and kidney and low activity in, for example, skeletal muscle. Mn superoxide dismutase activity was high in organs with high respiration, such as liver, kidney, and myocardium. Overall the Mn superoxide dismutase activity in organs was almost as high as the CuZn superoxide dismutase activity. The content of extracellular superoxide dismutase was, almost without exception, lower than the content of the other isoenzymes. The pattern of tissue distribution was distinctly different from those of CuZn superoxide dismutase and Mn superoxide dismutase. The tissue distribution of extracellular superoxide dismutase differed among species, but in general there was much in lungs and kidneys and little in skeletal muscle. In man, pig, sheep, cow, rabbit and mouse the overall tissue extracellular superoxide dismutase activities were similar to each other, whereas dog, cat and rat tissues contained distinctly less. There was no general correlation between the tissue extracellular superoxide dismutase activity of any of the various species and the variable plasma activity. The ratio between the plasma and the overall tissue activities was high, for some species over unity, providing further evidence for the notion that one role of extracellular superoxide dismutase is as a plasma protein.     

Glucose tolerance in aging obese (ob/ob) and lean mice

Examination of the glucose tolerance in younger (3 month) and older (6 month) obese mice revealed that most of their postinjection hyperglycemia arises from the disproportionately large glucose responses to the injection/bleeding procedures rather than from the added glucose. Simultaneous measurements of circulating glucagon, corticosterone and insulin indicated that simple differences in the levels of these hormones, in their circulating ratios, or in the magnitude of the hormone responses to stimulation did not fully account for the "stress"-induced hyperglycemia.     

Isolated adipocytes from growth hormone-treated obese (ob/ob) mice exhibit insulin resistance

The genetically obese (ob/ob) mouse is a useful model for the study of the diabetogenic action of growth hormone (GH), because treatment of these animals with GH results in decreased responsiveness of their adipose tissue to insulin in vitro. Studies of the mechanisms involved in GH-induced insulin resistance using isolated adipocytes of ob/ob mice have not been possible, however, because of their extreme fragility and the lack of an adequate system for the maintenance of these cells. This study describes a new method for the isolation of ob/ob mouse adipocytes. The isolated cells are stable, viable and metabolically responsive to insulin. In addition, these adipocytes have been maintained in primary culture, in serum-free medium, for up to 3 days. During culture, the cells exhibit large increases in 125I-hGH binding (10-20-fold) and porcine 125I-insulin binding (5-10-fold). The induction of insulin resistance by GH has also been demonstrated in these freshly isolated ob/ob mouse adipocytes. The studies to date indicate that the ob/ob mouse adipocyte system should provide a useful model for detailed studies of the cellular and molecular mechanisms of GH induced insulin resistance.     

Extracellular superoxide dismutase

The extracellular space is protected from oxidant stress by the antioxidant enzyme extracellular superoxide dismutase (EC-SOD), which is highly expressed in selected tissues including blood vessels, heart, lungs, kidney and placenta. EC-SOD contains a unique heparin-binding domain at its carboxy-terminus that establishes localization to the extracellular matrix where the enzyme scavenges superoxide anion. The EC-SOD heparin-binding domain can be removed by proteolytic cleavage, releasing active enzyme into the extracellular fluid. In addition to protecting against extracellular oxidative damage, EC-SOD, by scavenging superoxide, preserves nitric oxide bioactivity and facilitates hypoxia-induced gene expression. Loss of EC-SOD activity contributes to the pathogenesis of a number of diseases involving tissues with high levels of constitutive extracellular superoxide dismutase expression. A thorough understanding of the biological role of EC-SOD will be invaluable for developing novel therapies to prevent stress by extracellular oxidants.     

Effects of acute exercise over heart proteome from monogenic obese (ob/ob) mice

Exercise is recognized to prevent and attenuate several metabolic and cardiovascular disorders. Obesity is commonly related to cardiovascular diseases, frequently resulting in heart failure and death. To elucidate the effects of acute exercise in heart tissue from obese animals, 12‐week‐old C57BL6/J obese (ob/ob) and non‐obese (ob/OB) mice were submitted to a single bout of swimming and had their hearts analyzed by proteomic techniques. Mice were divided into three groups: control (ob/ob, n = 3; ob/OB, n = 3); a moderate intensity consisting of 20 min of swimming around 90% of Maximal Lactate Steady State (ob/ob, n = 3; ob/OB, n = 3), and a high intensity exercise performed as an incremental overload test (ob/ob, n = 3; ob/OB, n = 3). Obesity modulations were analyzed by comparing ob/ob and ob/OB control groups. Differential 2‐DE analysis revealed that single session of exercise was able to up‐regulate: myoglobin (ob/ob), aspartate aminotransferase (ob/OB) and zinc finger protein (ob/OB) and down‐regulate: nucleoside diphosphate kinase B (ob/OB), mitochondrial aconitase (ob/ob and ob/OB) and fatty acid binding protein (ob/ob). Zinc finger protein and α‐actin were up‐regulated by the effect of obesity on heart proteome. These data demonstrate the immediate response of metabolic and stress‐related proteins after exercise so as contractile protein by obesity modulation on heart proteome. J. Cell. Physiol. 228: 824–834, 2013. © 2012 Wiley Periodicals, Inc.     

Islet cell surface antibodies in genetically obese hyperglycaemic (ob/ob) mice

A quantitative method for circulating islet cell surface antibodies (ICSA), based on the binding of¹²⁵I-protein A to insulin-producing RINm5F cells, was used to evaluate ICSA in plasma of 4- to 40-week-old Aston obese hyperglycaemic (ob/ob) mice and normal control (+/+) mice. RINm5F cells bound 2502±l196 c.p.m.¹²⁵I-protein A per l0⁵ cells (mean±S.D.,n=54) after incubation with +/+ plasma. ICSA positive plasma (defined as¹²⁵I-protein A binding, mean±2 S.D. of +/+ plasma) was detected in 3 out of 54+/+ mice and 3 out of 54 ob/ob mice. ICSA were not observed in ob/ob mice before the onset of diabetes (7 weeks of age), but were detected at 9, 20 and 40 weeks. At 20 weeks¹²⁵I-protein A binding produced by ob/ob plasma was 35% greater than +/+ plasma (P<0.05). The low occurrence of ICSA in ob/ob mice (6%) suggests that factors other than ICSA are responsible for B-cell dysfunction and eventual islet degeneration observed in Aston ob/ob mice.     

Adrenalectomy and steroid treatment in obese (ob/ob) and diabetic (db/db) mice

Adrenalectomy in young obese (ob/ob) and the diabetic (db/db) mouse slowed body weight gain. Treatment of adrenalectomized ob/ob mice with cortisone or deoxycorticosterone acetate (DOCA) significantly increased weight gain in a dose-related manner. Cortisone had no effect on weight gain on lean mice and treatment with dehydroepiandrosterone sulfate was without effect on either ob/ob or lean mice. The increment in body weight of adrenalectomized ob/ob mice treated with corticosterone and DOCA was associated with an increase in body weight and an increase in food intake. When adrenalectomy was performed at twenty-three days of age (five days before weaning), animals carrying the (db/db) genotype remained lighter than their normal littermates. These data document the importance of the adrenal gland and its steroids for the development and maintenance of many features of the obese or diabetes mouse.     

Adrenalectomy increases norepinephrine turnover in brown adipose tissue of obese (ob/ob) mice

The hyperphagia and rapid body weight gain normally observed in young obese (ob/ob) mice were abolished by removal of their adrenal glands, whereas food intake and weight gain of lean mice were not significantly affected by adrenalectomy. Adrenalectomy lowered body energy density (kcal/g carcass) in obese mice more than could be attributed to reduced food intake per se, suggesting that their energy expenditure was also increased. In control obese mice, low stimulation of brown adipose tissue by the sympathetic nervous system, as indicated by the low fractional rates of norepinephrine (NE) turnover in their brown adipose tissue may have contributed to the reduced energy expenditure in these animals. Adrenalectomy increased the rates of NE turnover in brown adipose tissue of obese mice to rates nearly equal to those observed in lean mice without affecting NE turnover in this tissue of lean mice. Likewise, removal of the adrenals normalized the low rates of NE turnover in hearts of obese mice without affecting lean mice. Rates of NE turnover in two other organs, white adipose tissue and pancreas, of control and adrenalectomized obese mice were similar to rates observed in lean counterparts. The adrenal may thus contribute to both the hyperphagia and the low energy expenditure by brown adipose tissue that together cause gross obesity in ob/ob mice.     

Decreased triiodothyronine binding to isolated nuclei from livers of preobese and obese (ob/ob) mice

L-Triiodothyronine (T3) binding to hepatic nuclei from (ob/ob) mice at different ages was examined and compared with that of lean controls. Results showed a significant reduction in T3 binding in liver nuclei of obese mice at all ages studied. The preobese mice at 2 weeks of age had 27.9% fewer receptor sites/mg DNA compared to lean controls, receptor concentration further decreased to 67.7% at 18 weeks of age. Data presented here demonstrates that the impaired triiodothyronine (T3) binding to hepatic nuclei present in older (ob/ob) obese mice is an antecedent to the obesity. This report also helps to explain the poor thermoregulation and low oxygen consumption present during the preobese phase of the postnatal development of these animals.     

Effects of insulin on lipolysis and lipogenesis in adipocytes from genetically obese (ob/ob) mice.

A method for the preparation of isolated adipocytes from obese mice is described. Similar yields of adipocytes (50--60%), as judged by several criteria, are obtained from obese mice and lean controls. Few fat-globules and no free nuclei were observed in cell preparations, which are metabolically active, respond to hormonal control and appear to be representative of intact adipose tissue. Noradrenaline-stimulated lipolysis was inhibited by insulin, equally in adipocytes from lean and obese mice. Inhibition in obese cells required exogenous glucose, and the insulin dose--response curve was shifted to the right. Basal lipogenesis from glucose was higher in adipocytes from obese mice, and the stimulatory effect of insulin was greater in cells from obese mice compared with lean controls. A rightward shift in the insulin dose--response curve was again observed with cells from obese animals. This suggests that adipose tissue from obese mice is insulin-sensitive at the high blood insulin concentrations found in vivo. The resistance of obese mice to the hypoglycaemic effect of exogenous insulin and their impaired tolerance to glucose loading appear to be associated with an impaired insulin response by muscle rather than by adipose tissue.     

Glucoregulatory effects of bombesin in lean and genetically obese hyperglycaemic (ob/ob) mice

1. Plasma glucose and insulin responses to bombesin were examined in 12-15-week-old 12 hr fasted lean and genetically obese hyperglycaemic (ob/ob) mice. 2. Bombesin (1 mg/kg ip) produced a prompt but transient increase of plasma insulin in lean mice (maximum increase of 50% at 5 min), and a more slowly generated but protracted insulin response in ob/ob mice (maximum increase of 80% at 30 min). Plasma glucose concentrations of both groups of mice were increased by bombesin (maximum increases of 40 and 48% respectively in lean and ob/ob mice at 15 min). 3. When administered with glucose (2 g/kg ip), bombesin (1 mg/kg ip) rapidly increased insulin concentrations of lean and ob/ob mice (maximum increases of 39 and 63% respectively at 5 min). Bombesin did not significantly alter the rise of plasma glucose after exogenous glucose administration to these mice. 4. The results indicate that bombesin exerts an insulin-releasing effect in lean and ob/ob mice. The greater insulin-releasing effect in ob/ob mice renders bombesin a possible component of the overactive entero-insular axis in the ob/ob mutant, especially if it acts within the islets as a neurotransmitter or paracrine agent.     

Glucagon-like peptide-1 and the entero-insular axis in obese hyperglycaemic (ob/ob) mice

The intestines of obese hyperglycaemic (ob/ob) mice contain greatly increased amounts of glucagon-like immunoreactive peptides. To investigate their role in the increased activity of the entero-insular axis of these mice, the insulin-releasing effect of glucagon-like peptide-1 (GLP-1) was examined in 24 hour fasted 12-15 weeks old ob/ob mice under conditions of basal and elevated glycaemia. Compared with glucagon (100 micrograms/kg ip), which produce an approximately 3-fold increase in basal plasma glucose and insulin concentrations, GLP-1 (100 micrograms/kg ip) produce a very small (less than 1 fold) increase in plasma insulin, with no significant change in plasma glucose. The insulin-releasing effect of glucagon, but not GLP-1 was increased by administration in combination with glucose (2 g/kg ip). The results indicate that GLP-1, which exhibits considerable sequence homology with glucagon, exerts only a weak insulin-releasing effect without a significant hyperglycaemic effect in ob/ob mice. Thus GLP-1 is unlikely to be an important endocrine component of the two over-active entero-insular axis in ob/ob mice.     

Resistance to hepatic action of vasopressin in genetically obese (ob/ob) mice.

1. Fatty acid synthesis, measured in the perfused liver of genetically obese (ob/ob) mice with 3H2O or [14C]actate, did not show the inhibition by [8-arginine]vasopressin (antidiuretic hormone) that is observed in livers from normal mice. 2. Hepatic glycogen breakdown in obese mice was stimuulated by vasopressin, but not as extensively as in lean mice. 3. If obese mice received a restricted amount of food, then fatty acid synthesis still did not respond to vasopressin, but glycogen breakdown was fully stimulated. 4. Cholesterol synthesis was not inhibited by vasopressin in livers from obese mice. 5. Vasopressin inhibited fatty acid synthesis in intact lean mice, but not in obese animals. 6. These results suggest that genetic obesity could be due to an inborn error within the mechanisms (other than adenylate cyclase) which mediate responses to extracellular effectors.     

Dietary copper supplementation increases the catecholamine levels in genetically obese (ob/ob) mice

The interactive relationship between Cu deficiency and depressed synthesis of certain neurotransmitters has been recognized. To investigate the effects of dietary Cu supplementation on the catecholamine levels in genetically obese mice, male obese (ob/ob) mice and their lean (+/?) counterparts were administered either a control diet (4.0 mg/kg) or a Cu-supplemented diet (50 mg/kg) for 4 wk. The ob/ob mice that were fed a control diet showed lower liver and higher plasma levels of Cu. Depressed levels of plasma and brain catecholamines were also found in ob/ob mice that were fed the control diet. The ob/ob mice that received a Cu-supplemented diet showed significant increases in the levels of catecholamine in the plasma and brain. This study showed that catecholamine levels in ob/ob mice can be increased by dietary Cu supplementation. However, the interaction between Cu and sympathetic nervous activity in obesity was not elucidated in this study.     

Unimpaired thermogenic response to noradrenaline in genetic (ob/ob) and hypothalamic (MSG) obese mice

The thermogenic response to noradrenaline administra-tion was investigated at 25° in two models of obese mice (genetic ob/ob obesity of the QEC strain and monosodium-glutamate-induced obesity) and in their respective lean littermates. Subcutaneous injections of a low dose of noradrenaline (I00 g/kg body wt.) eJevated metabolic rate by about 3096 in both obese models but not in their respective lean counterparts. In contrast, the increase in metabolic rate after injections of a high dose of noradrenaline (600 g/kg body wt.) was of a similar magnitude in both lean and obese animals: metabolic rate was increased by 70–80%. These results indicate that the overall whole body thermogenic capacity is unimpaired at room temperature in this QEC strain of ob/ob mice and in the hypothalamic damaged obese mice. Obesity in these models is therefore not associated with a reduced ability to respond to noradrenaline but could rather be due to a failure to release noradrenaline.     

Effect of metformin on nitric oxide synthase in genetically obese (ob/ob) mice.

Genetically obese (ob/ob) mice were employed for the study of the effect of metformin on activity and expression of nitric oxide synthase (NOS ) in vitro and in vivo. For in vitro analysis, mouse liver extracts were used. For the in vivo study, (ob/ob) and their control litter mates (ob/c) mice were injected with specified amounts of metformin and the expression of NOS in the adipose tissue and hypothalamus was measured by Western blotting. Results show that metformin exhibited a biphasic effect on NOS activity in vitro. Expression of metformin was differentially altered in the hypothalamus and adipose tissues of the normal and ob/ob animals that were treated with metformin. Further, a significant decrease in food intake occurred in the (ob/ob) mice that received metformin. This decrease in food intake was not accompanied by changes in serum glucose. At inhibitory concentrations, hypothalamic NOS expression changes differentially in normal and ob/ob mice. In normal mice, metformin stimulated NOS expression, while in ob/ob mice there was an inhibition. NOS expression increased in brown adipose tissue of metformin treated control mice, while no such increase was observed in ob/ob mice. No effect of metformin was observed in white adipose tissue of control or obese mice. Thus, metformin may produce anorectic effects through modulation of NOS.