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.     

Effects of cold acclimation and fasting on thyroxine 5'-deiodinase in brown adipose tissue of ob/ob mice.

Gradual acclimation to mild cold for 6 weeks increases the total activity of thyroxine 5'-deiodinase in brown adipose tissue (BAT) of genetically obese (ob/ob) mice to a level greater than that in similarly acclimated lean mice. This increase is largely due to the growth of the BAT in the ob/ob mouse, because specific activity of the enzyme is only slightly increased. In similarly cold-acclimated lean mice, the specific activity of thyroxine 5'-deiodinase was not altered. BAT mitochondrial GDP binding increased to the same high level in the gradually cold-acclimated ob/ob mouse as in cold-acclimated lean mice. We conclude that the growth and maintenance of BAT in the cold-acclimated ob/ob mouse, as in the cold-acclimated lean mouse, does not require greatly increased activity of thyroxine 5'-deiodinase. Fasting for 48 hr did not alter thyroxine 5'-deiodinase activity of BAT in either lean or ob/ob mice. The fasting-induced increase in activity seen by others in lean mice is probably due to thermoregulatory stimulation of BAT occasioned by the low environmental temperature at which the fasting occurred.     

Adrenalectomy in genetically obese ob/ob and db/db mice increases the proton conductance pathway

Adrenalectomy (ADX) prevents the excessive weight gain in the genetically obese ob/ob and db/db mice. To test the possibility that this results from increased energy expenditure due to increased thermogenesis in brown adipose tissue (BAT), we measured GDP binding to mitochondria from interscapular brown adipose tissue (BAT) in db/db and ob/ob mice and their lean controls after adrenalectomy, with and without corticosterone replacement. Both the vehicle treated and corticosterone treated db/db and ob/ob mice had lower body weights than the sham-operated mice GDP binding to mitochondria from IBAT was significantly lower in both the db/db and ob/ob mice than in their lean controls. Adrenalectomy significantly increased GDP binding in all mice compared to the respective sham-operated mice, but, the percentage increase was always greater in the db/db and ob/ob mice. Corticosterone treatment of adrenalectomized db/db, ob/ob or lean mice lowered GDP binding to sham levels. Our data confirm previous findings that adrenalectomy results in increased GDP binding to mitochondria from IBAT. Injections of corticosterone into adrenalectomized mice results in a decrease in GDP binding to values which are similar to values in sham-operated mice. Thus adrenalectomy may inhibit the development of obesity by increasing the thermic activity in IBAT.     

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.     

Desaturation of stearic acid by liver and adipose tissue from obese-hyperglycaemic mice (ob/ob).

Stearic acid desaturase activity was assayed in preparations from perigenital adipose tissue and liver from lean and genetically obese female mice (ob/ob). The total activity in the perigenital adipose tissue from obese mice was threefold greater than in the tissue from lean mice, but per g of adipose tissue the activity was twofold greater in tissue from lean mice. In liver, the activity in obese mice was elevated at 8 weeks of age, remained elevated up to 24 weeks and then decreased by half at 48 weeks, but at all ages was higher than that in lean mice. The decrease in desaturase activity of liver from obese mice at 48 weeks corresponded to a change in the fatty acid composition of liver lipids toward that found in lean mice. Whereas in adipose tissue much of the increased enzyme activity may be due to tissue hyperplasia, in liver it is mainly an increased activity per cell.     

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.     

Obesity modulates the expression of haptoglobin in the white adipose tissue via TNFalpha.

Increase in adipose mass results in obesity and modulation of several factors in white adipose tissue (WAT). Two important examples are tumor necrosis factor alpha (TNFalpha) and leptin, both of which are upregulated in adipose tissue in obesity. In order to isolate genes differentially expressed in the WAT of genetically obese db/db mice compared to their lean littermates, we performed RNA fingerprinting and identified haptoglobin (Hp), which is significantly upregulated in the obese animals. Hp is a glycoprotein induced by a number of cytokines, LPS (Lipopolysaccharide), and more generally by inflammation. A significant upregulation of WAT Hp expression was also evident in several experimental obese models including the yellow agouti (/) A(y), ob/ob and goldthioglucose-treated mice (10-, 8-, and 7-fold, respectively). To identify the potential signals for an increase in Hp expression in obesity, we examined leptin and TNFalpha in vivo. Wild type animals treated with recombinant leptin did not show any alteration in WAT Hp expression compared to controls that were food restricted to the level of intake of the treated animals. On the other hand, Hp expression was induced in mice transgenically expressing TNFalpha in adipose tissue. Finally, a significant downregulation of WAT Hp mRNA was observed in ob/ob mice deficient in TNFalpha function, when compared to the ob/ob controls. These results demonstrate that haptoglobin expression in WAT is increased in obesity in rodents and TNFalpha is an important signal for this regulation.     

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.     

Effects of cold acclimation on the activity of lipoprotein lipase in adipose tissues of genetically obese Zucker rats

The activity of lipoprotein lipase (LPL) was studied in interscapilar brown adipose tissue (BAT), epididymal white adipose tissue (WAT) and in the heart of lean and obese adult Zucker rats maintained at 22 degrees C or adapted to cold (10 degrees C). In WAT the specific activity per gram of tissue was lower in obese than in lean rats but the total activity within the tissue was three-fold higher. Cold acclimation did not modify total activity in either lean or obese rats. In BAT, but not in the heart, both specific and total activities were lower in obese than in lean animals. They were enhanced in both tissues following cold acclimation. Six-hour fasting led to a decrease in specific activity in WAT of lean rats but had no effect in obese animals; an increase was observed in BAT and heart of both genotypes. Insulin administration has no effect on activities in WAT in either 22 or 10 degrees C adapted obese rats. Norepinephrine administration stimulates LPL activity in BAT and heart of all groups. It is concluded that the lack of development of obesity previously observed in obese rats following cold acclimation is not due to a decreased capacity of lipid uptake by WAT. It might in part be due to an increased lipid oxidation in BAT.     

Adenylate cyclase activity in brown adipose tissue of the genetically obese (ob/ob) mouse

The activation of brown adipose tissue adenylate cyclase by catecholamines was studied in genetically obese (ob/ob) and lean mice. In obese mice, the maximum activation of the enzyme by several beta-adrenergic agonists was only two-thirds that in lean mice and, as an activator, noradrenaline was only one-eighth as potent. The adenylate cyclase was also less responsive to guanine nucleotides. In these respects, the defect in catecholamine-stimulated adenylate cyclase was similar in both white and brown adipose tissue of the obese mouse. The enzyme in brown adipose tissue differed from that in white adipose tissue in its sensitivity to other beta-adrenergic agonists and in its requirement for Mg2+. It is suggested that this abnormal catecholamine-activated adenylate cyclase in brown adipose tissue may be relate to the thermoregulatory defect of the obese mouse and hence may contribute to the obesity syndrome.     

Attenuated cold-induced increase in mRNA for uncoupling protein in brown adipose tissue of obese (ob/ob) mice

The level of mRNA for uncoupling protein was measured in brown adipose tissue of young (8-10 weeks) and old (11 months) lean and ob/ob mice using a cDNA clone constructed previously. The level of poly(A)+ RNA was also measured using an oligo(dT)18 probe. Mice were kept at 28 degrees C or exposed to 14 degrees C for 12 h. The level of mRNA for uncoupling protein was normal in brown adipose tissue of younger obese mice but reduced in brown adipose tissue of old obese mice. The cold-induced absolute increase in uncoupling protein mRNA was smaller in obese mice, regardless of age. It is concluded that the known attenuation of the acute thermogenic response of brown adipose tissue of the ob/ob mouse to cold is accompanied by a similar attenuation of the initiation of the trophic response. It is likely, however, that these defects are secondary to the chronic reduction in sympathetic nervous system activity in brown adipose tissue of the ob/ob mouse, which results in a functional atrophy of the tissue.     

Transplantation of wild-type white adipose tissue normalizes metabolic, immune and inflammatory alterations in leptin-deficient ob/ob mice

Leptin-deficient ob/ob mice exhibit several metabolic and immune abnormalities, including thymus atrophy and markedly reduced inflammatory responses. We evaluated whether transplantation of wild-type (WT) white adipose tissue (WAT) into ob/ob mice could mimic the effect of recombinant leptin administration in normalizing metabolic, immune and inflammatory abnormalities. Female ob/ob mice received a subcutaneous transplantation of WAT obtained from WT littermates. A separate group of ob/ob mice was sham-operated. Despite raising leptin levels to only 15% of those observed in WT mice, WAT transplantation normalized metabolic abnormalities (glycemia, ALT, liver weight) in ob/ob mice and prevented further body weight gain. The transplanted group demonstrated normalization of thymus and spleen cellularity, thymocyte subpopulations and rates of thymocyte apoptosis. In the model of dextran sulfate sodium-induced colitis, WAT transplantation restored inflammation to levels equivalent to those of WT mice. Colonic production of IL-6 and MIP-2 was markedly reduced in the non-transplanted ob/ob group compared to transplanted ob/ob and WT mice. Our data indicate that WAT transplantation is an effective way to normalize metabolic as well as immune and inflammatory parameters in ob/ob mice. The threshold of leptin sufficient to normalize metabolic, immune and inflammatory function is significantly lower than levels present in lean WT mice. Finally, leptin derived exclusively from WAT is sufficient to normalize metabolic, immune and inflammatory parameters in ob/ob mice.     

Genetically obese C57BL/6 ob/ob mice respond normally to sympathomimetic compounds

The suggestion that defective thermoregulatory thermogenesis in the genetically obese (ob/ob) mouse is due to a low thermic response to noradrenaline has been investigated using both noradrenaline and the longer-acting sympathomimetic compounds, ephedrine and BRL 26830A. Below thermoneutrality (23.5°C) the metabolic rate of obese mice was lower than that of their lean littermates, whereas at a thermoneutral temperature (31°C) the metabolic rate of the obese nice was as high as that of lean mice. This confirms the view that the ob/ob mouse has defective thermoregulatory thermogenesis. However, in C57BL/6 mice, this defect is not due to a failure to respond to noradrenaline, because at 31°C the maximum thermic effects of noradrenaline, ephedrine and BRL 26830A were as high in obese as in lean mice and at 23.5°C they were higher in obese than in lean mice. Furthermore, the response of brown adipose tissue to β-adrenoceptor stimulation appears normal since noradrenaline caused a normal rise in brown adipose tissue temperature, and treatment with noradrenaline or BRL 26830A $\text{in}$$\text{vivo}$ caused a normal increase in GDP binding by brown adipose tissue mtiochondria. At 31°C propranolol depressed metabolic rate equally in lean and obese C57BL/6 mice, whereas at 23.5°C it depressed metabolic rate more in lean than obese mice. In contrast to C57BL/6 mice, Aston ob/ob mice showed a reduced thermic response to noradrenaline. These results suggest that defective thermoregulatory thermogenesis in the ob/ob mouse is primarily due to a reduced ability to raise sympathetic tone, but in some strains an additional failure in the thermic response to noradrenaline may develop.     

Continuous flow microcalorimetric measurement of heat production in white adipose tissue from obese (ob/ob) mice and their lean littermates

Heat production, free fatty acid and glycerol release from white adipose tissue fat pads from obese (ob/ob) mice and their lean littermates are determined. Heat production was significantly lower in obese mice compared to lean mice when expressed on wet weight basis but not when expressed on DNA basis. Noradrenaline significantly increased the heat production in fat pads from both groups of animals. However, the increase in heat production due to noradrenaline addition in fat pads from lean mice was significantly higher than in fat pads from obese mice. The release of free fatty acids and glycerol before incubation with noradrenaline was similar from fat pads from both groups of animals. Addition of noradrenaline to the fat pads increased the release of free fatty acids and glycerol in both groups of animals, but the increase was significantly larger from fat pads from lean mice. In the absence of noradrenaline the free fatty acid/glycerol ratio (mol/mol) in the effluent was 7.9:1 and 4.8:1 for lean mice and obese mice, respectively. In the presence of noradrenaline the ratio decreased to 3:1 for both groups of animals.     

Reduced levels of peroxisomal enzymes in the kidney of the genetically obese (ob/ob) mouse. Contrast with liver

Kidney post-nuclear supernatants from genetically lean and obese mice were subjected to subcellular fractionation by dual centrifugation through sucrose gradients in B XIV zonal rotors. Considerable purification of peroxisomes was achieved which allowed the demonstration of acyl-CoA beta-oxidation enzymes and carnitine acyltransferases in these organelles. Comparison of kidney peroxisome-enriched fractions from obese and lean mice indicated a likely relative depression in beta-oxidation enzymes in the obese animal. Measurement of catalase, acyl-CoA oxidase and carnitine octanoyltransferase in whole homogenate of liver and kidney of obese and lean mice revealed significantly reduced levels (to approximately 2/3) of these peroxisomal enzymes in the kidney of ob/ob mice. In contrast the specific activity of catalase and acyl-CoA oxidase was significantly raised in the liver of obese mice.     

Alterations in Oral [1-14C] 18:1n-9 Distribution in Lean Wild-Type and Genetically Obese (ob/ob) Mice

Obesity may result from altered fatty acid (FA) disposal. Altered FA distribution in obese individuals is poorly understood. Lean wild-type C57BL/6J and obese C57BL/6J^ob/ob mice received an oral dose of [1-¹⁴C]18:1n-9 (oleic acid), and the radioactivity in tissues was evaluated at various time points. The ¹⁴C concentration decreased rapidly in gastrointestinal tract but gradually increased and peaked at 96 h in adipose tissue, muscle and skin in lean mice. The ¹⁴C concentration was constant in adipose tissue and muscle of obese mice from 4h to 168h. ¹⁴C-label content in adipose tissue was significantly affected by genotype, whereas muscle ¹⁴C-label content was affected by genotype, time and the interaction between genotype and time. There was higher total ¹⁴C retention (47.7%) in obese mice than in lean mice (9.0%) at 168 h (P<0.05). The ¹⁴C concentrations in the soleus and gastrocnemius muscle were higher in obese mice than in lean mice (P<0.05). Perirenal adipose tissue contained the highest ¹⁴C content in lean mice, whereas subcutaneous adipose tissue (SAT) had the highest ¹⁴C content and accounted for the largest proportion of total radioactivity among fat depots in obese mice. More lipid radioactivity was recovered as TAG in SAT from obese mice than from lean mice (P<0.05). Gene expression suggested acyl CoA binding protein and fatty acid binding protein are important for FA distribution in adipose tissue and muscle. The FA distribution in major tissues was altered in ob/ob mice, perhaps contributing to obesity. Understanding the disparity in FA disposal between lean and obese mice may reveal novel targets for the treatment and prevention of obesity.     

Effect of clofibrate on malic enzyme and leptin mRNAs level in rat brown and white adipose tissue.

Two previous studies have reported contradictory results regarding the effect of fibrates treatment on obese (ob) gene expression in rodents. The purpose of the present study was to reinvestigate this issue. We examined the effect of clofibrate (fibrate derivative) administration for 14 days to rats on malic enzyme (as an adequate control of fibrates action) and leptin mRNAs level in the white and brown adipose tissues (WAT and BAT, respectively). The malic enzyme activity and malic enzyme mRNA level in white adipose tissue increased significantly after clofibrate feeding. In brown adipose tissue, the drug treatment resulted in depression of malic enzyme activity and malic enzyme mRNA level. Under the same conditions, leptin mRNA level did not change in these tissues. The results presented in this paper provide further evidence that the clofibrate (activator of peroxisome proliferator activated receptor alpha), feeding is without effect on ob gene expression in rat white and brown adipose tissue. Furthermore, the present study demonstrates that clofibrate causes opposite effects on malic enzyme gene expression in WAT (up-regulation) and BAT (down-regulation).     

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.     

Leptin deficiency leads to the regulation of kinin receptors expression in mice

Kinins are vasoactive and pro-inflammatory peptides generated by the cleavage of the kininogen by kallikreins. Two kinin receptors have been described and denominated B1 and B2. Obesity frequently accompanies other pathologies, such as diabetes and hypertention. The clustering of these pathologies is usually known as "metabolic syndrome". Mice lacking leptin gene (ob/ob) are severely obese and hyperphagic. Using quantitative RT-PCR analysis of B1 and B2 mRNAs expression, we described for the first time a correlation between the kallikrein-kinin system (KKS) and severe obesity in mice. The ob/ob mice presented lower expression of B2 mRNA in the white adipose tissue (WAT) and hypothalamus, both primary sites for neuroendocrine regulation of the energetic metabolism. B1 mRNA, however, is overexpressed in these tissues of ob/ob mice. An upregulation of the B1 mRNA has also been seen in liver, abdominal aorta and stomach fundus. However, different from the lean mice, the expression of the B1 mRNA in brown adipose tissue (BAT) and heart is completely abolished. Our data show that kinin receptors are differently modulated in distinct tissues in obesity. These findings suggest a connection between the KKS and obesity, and suggest that kinin receptors could be involved in the ethiopathogenesis of the metabolic syndrome.