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.     

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.     

Mitochondrial Ca2+ transport in lean and genetically obese (ob/ob) mice.

Isolated mitochondria from liver or brown adipose tissue of obese ob/ob mice demonstrated increased rates of Ca2+ uptake and release compared with those of lean mice. This enhanced transport activity was not found in mitochondria from kidney or skeletal muscle. Respiration-induced membrane potential was the same in mitochondria from lean and ob/ob mice. It is therefore concluded that the increased Ca2+ uptake rates reflect an activation of the Ca2+ uniporter rather than a change in the electrophoretic driving force. As mitochondria from pre-obese ob/ob mice did not show elevated rates of Ca2+ transport, the activated transport in the obese animals was thus a consequence of the state of obesity rather than being a direct effect of the ob/ob genotype. It is suggested that the enhanced activity of the Ca2+-transport pathways in liver and brown adipose tissue may alter metabolic functions in these tissues by modifying cytoplasmic or intramitochondrial Ca2+ concentrations.     

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.     

Zinc attenuation of GDP binding to brown adipocytes mitochondria in genetically obese (ob/ob) mice

In this study, we investigate the in vitro effect of zinc addition on guanosine diphosphate (GDP) binding to mitochondria in brown adipocytes of genetically obese (ob/ob) mice. Interscapular brown adipocytes of male mice (obese; lean) at 4 and 12 wk of age were incubated with 0, 50, 100, or 200 μM zinc sulfate. Mitochondria were then isolated and their GDP binding capacities were measured. The GDP-binding capacities of ob/ob mice were lower than lean mice, with or without zinc addition, in both age groups (p<0.05). Zinc addition did not have any significant effect on GDP binding in lean mice. GDP binding decreased with increasing zinc addition in ob/ob mice, and this attenuation was more predominant in 12-wk old ob/ob mice. Moreover, we found that high magnesium addition (5 mM) increased GDP binding in lean mice, but this effect was not significant in ob/ob mice. This study reveals that brown adipose tissue thermogenesis in ob/ob mice could be greatly attenuated by zinc addition, suggesting that zinc may play a regulatory role in obesity.     

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.     

Hypothalamic adrenergic receptor changes in the metabolic syndrome of genetically obese (ob/ob) mice

The genetically, seasonally, and diet-induced obese, glucose-intolerant states in rodents, including ob/ob mice, have each been associated with elevated hypothalamic levels of norepinephrine (NE). With the use of quantitative autoradiography on brain slices of 6-wk-old obese (ob/ob) and lean mice, the adrenergic receptor populations in several hypothalamic nuclei were examined. The binding of [(125)I]iodocyanopindolol to beta(1)- and beta(2)-adrenergic receptors in ob/ob mice was significantly increased in the paraventricular hypothalamic nucleus (PVN) by 30 and 38%, in the ventromedial hypothalamus (VMH) by 23 and 72%, and in the lateral hypothalamus (LH) by 10 and 15%, respectively, relative to lean controls. The binding of [(125)I]iodo-4-hydroxyphenyl-ethyl-aminomethyl-tetralone to alpha(1)-adrenergic receptors was also significantly increased in the PVN (26%), VMH (67%), and LH (21%) of ob/ob mice. In contrast, the binding of [(125)I]paraiodoclonidine to alpha(2)-adrenergic receptors in ob/ob mice was significantly decreased in the VMH (38%) and the dorsomedial hypothalamus (17%) relative to lean controls. This decrease was evident in the alpha(2A)- but not the alpha(2BC)-receptor subtype. Scatchard analysis confirmed this decreased density of alpha(2)-receptors in ob/ob mice. Together with earlier studies, these changes in hypothalamic adrenergic receptors support a role for increased hypothalamic NE activity in the development of the metabolic syndrome of ob/ob mice.     

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.     

Anorectic effect of fenfluramine, cholecystokinin and neurotensin in genetically obese (ob/ob) mice

To investigate the satiety defect of hyperphagic genetically obese (ob/ob) mice, acute feeding responses to three differently acting anorectic agents were determined in 7-9 weeks old lean (+/+) and ob/ob mice habituated to a restricted (0900-1230 hr) daily feeding routine. Fenfluramine (10 mg/kg), cholecystokinin (100 U/kg) and neurotensin (500 micrograms/kg), administered intraperitoneally 15 min before feeding, each produced a rapid but transient suppression of food consumption in ob/ob mice, similar to lean controls. The results suggest that neural satiety mechanisms triggered via serotoninergic pathways (fenfluramine), vagal afferents (cholecystokinin) and the hypothalamic paraventricular nucleus (neurotensin) are functional in ob/ob mice, supporting the view that the satiety defect of ob/ob mice resides outside of the nervous system.     

Hepatic delta 6-desaturase activity in lean and genetically obese ob/ob mice.

Hepatic delta 6-desaturase activity is primarily located in the mitochondrial fraction in mice. Both delta 6- and delta 5-desaturase activities are increased in the liver of young (6-week-old) obese mice. The increase in hepatic delta 6-desaturase activity in obese mice does not occur until weaning. Neither restriction of food intake nor hyperinsulinaemia normalize hepatic delta 6-desaturase activity of obese mice. Both cold acclimation and tri-iodothyronine (30 micrograms/day per kg) decreased hepatic delta 6-desaturase activity of obese mice to levels observed in lean mice, whereas the increase in activity in obese mice was still maintained after the induction of hypothyroidism.     

Arrhenius plot characteristics of adipocyte-plasma-membrane adenylate cyclase activity in lean and genetically obese (ob/ob) mice

Arrhenius plots of fluoride- and guanine-nucleotide-stimulated adenylate cyclase activity were linear in adipocyte plasma membranes from lean and obese (ob/ob) mice . Arrhenius plots of isoprenaline-stimulated adenylate cyclase activity in hepatic plasma membranes biphasic in both groups. The results were biphasic in membranes from Jean mice but linear in membranes from obese mice. In contrast, Arrhenius plots of glucagon-stimulated adenylate cyclase activity in hepatic plasma membranes were biphasic in both groups. The results suggest that the coupling between the -receptor and the regulatory unit of adenylate cyclase, which has been observed to be defective in adipocyte plasma membranes from obese mice, is influenced by a different lipid environment in membranes from obese animals.     

Elevated hepatic mitochondrial and peroxisomal oxidative capacities in fed and starved adult obese (ob/ob) mice.

Hepatic mitochondrial and peroxisomal oxidative capacities were studied in young (4-5 weeks old) and adult (6-9 months old) lean and obese ob/ob mice that were fed or starved for 24 or 48 h. The adult obese mice showed elevated capacity for mitochondrial oxidation (ng-atoms of O consumed/min per mg of protein) of lipid and non-lipid substrates, with the exception of pyruvate + malate, and elevated activities of citrate synthase and total carnitine palmitoyltransferase. Oxidative rates and enzyme activities were not affected by starvation of lean or obese mice, and both males and females responded similarly. Peroxisomal palmitoyl-CoA oxidation (nmol/min per mg of peroxisomal protein) was also increased in livers of adult obese mice and did not change with starvation. In young mice, hepatic mitochondrial and peroxisomal oxidative capacities in lean and obese mice were comparable. The increased mitochondrial and peroxisomal oxidative capacities appear to develop with maturation in obese ob/ob mice.     

Developmental changes in fatty acid synthesis in interscapular brown adipose tissue of lean and genetically obese (ob/ob) mice.

Fatty acid synthesis was measured in vivo with 3H2O in interscapular brown adipose tissue of lean and genetically obese (ob/ob) mice. At 26 days of age, before the development of hyperphagia, synthesis in brown adipose tissue was higher in the obese than in the lean mice; synthesis was also elevated in the liver, white adipose tissue and carcass of the obese mice. At 8 weeks of age, when hyperphagia was well established, synthesis remained elevated in all tissues of the obese mice, with the exception of brown adipose tissue. Elevated synthesis rates were not apparent in brown adipose tissue of the obese mice at 14 days of age, nor at 35 days of age. These results demonstrate that brown adipose tissue in ob/ob mice has a transitory hyperlipogenesis at, and just after, weaning on to a low-fat/high-carbohydrate diet. Once hyperphagia has developed, by week 5 of life, brown adipose tissue is the only major lipogenic tissue in the obese mice not to exhibit elevated rates of fatty acid synthesis; this suggests that insulin resistance develops much more rapidly in brown adipose tissue than in other lipogenic tissues of the ob/ob mouse.     

Association of monoamine oxidase and malate dehydrogenase with liver peroxisomes of genetically obese (ob/ob and db/db) mice.

1. Liver post-nuclear supernatants (PNS) from genetically obese (ob/ob and db/db), lean (+/?), and albino mice were fractionated by dual centrifugation in B-XIV zonal rotors and subcellular fractions were analysed by marker-enzyme estimations and by electron microscopy. 2. Rate-dependent banding of PNS yielded a peroxisome-enriched region (PER) well-separated from mitochondria. 3. Density-dependent banding of PER in ob/ob and db/db mice only, yielded purified peroxisomes which were associated with malate dehydrogenase (cytosolic) and monoamine oxidase. 4. Markers for the mitochondrial matrix, intermembrane space and inner membrane compartments were absent from the peroxisomes. 5. The experimental results are interpreted as indicating that peroxisomes of genetically obese mice are either altered so that protein import is imprecise or so that their attachment to mitochondria is more extensive.     

Influence of restricted food intake on brown adipose tissue function in genetically obese mice (genotype, ob/ob)

Measurements were made of cytochrome c oxidase activity and the GDP-binding capacity of mitochondria in brown adipose tissue of genetically obese mice and wild-type siblings, to estimate the thermogenic capacity of the tissue. The binding capacity was decreased in ad libitum fed obese animals compared with wild-type animals. Limited feeding of obese animals to restrict their body weight caused a large increase in the binding capacity of the tissue, which was greater than that in wild-type animals fed either ad limitum or on a limited diet. The decreased binding capacity of brown adipose tissue mitochondria in obese mice appears to be a consequence of ad libitum feeding and therefore not a cause of the obesity. Limit feeding of obese animals also corrected their characteristic hypothermia at low ambient temperature. The large increase in the thermogenic capacity of brown adipose tissue in obese animals, induced by limited feeding, may account for the vital improvement of their thermoregulation. However, close similarities were found between obesity hypothermia and hypothermia induced in wild-type animals by restraint. It is suggested that changes in posture caused by obesity, resulting in increased loss of body heat, may be important in the development of obesity hypothermia. Obese animals fed less than wild-type grained more weight than wild-type animals, indicating that the high thermogenic capacity of their brown adipose tissue did not function to regulate their calorie intake.     

Zinc supplementation on serum levels and hepatic conversion of thyroid hormones in obese (ob/ob) mice

The supplemental effects of zinc on thyroid status in obese (ob/ob) mice were studied. Four-week-old obese mice and their lean controls were fed either a basal diet or a zinc-supplemented diet (200 mg/kg diet) for 8 wk. Following the 8-wk basal diet, obese mice had lower serum T4 values, as well as hepatic T4 and T3 values, than lean mice (p < 0.05). A significant decrease in hepatic 5′-deiodinase activity was also observed in obese mice. Dietary zinc supplementation significantly reduced serum T4 levels in both the obese and lean mice. However, the zinc-supplemented effects on diminishing hepatic T4 and T3 values, as well as on 5′-deiodinase activities, were found only in obese mice (p < 0.05). Furthermore, the 5′-deiodinase activities in hepatic microsomal pellets after incubation with various zinc concentrations (0.5, 1.0, and 2.5 mM) were also examined. The 5′-deiodinase activities, in hepatic samples from all mice, were significantly attenuated by zinc treatments. However, this effect was more predominant in obese mice following the addition of 0.5 mM zinc. This study suggests that a lower hepatic 5′-deiodinase activity, resulting from a higher zinc level, might be related to abnormal energy metabolism in theob/ob mice.     

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.