Membrane fluidity and adenylate cyclase activity in genetically obese mice

Adipocyte plasma membranes of genetically obese $\text{ob}\text{ob}$ mice are more fluid than their lean littermates but the fluidity was normalised in mice maintained at high environmental temperatures. The defective response of adenylate cyclase to isoproterenol was improved after normalisation of membrane fluidity. No major changes in the phospholipid composition of $\text{ob}\text{ob}$ membranes were detected.     

Fatty acid utilization and purine nucleotide binding in brown adipose tissue of genetically obese (ob/ob) mice

The activities of the main enzymes involved in fatty acid utilization i.e. palmitoyl CoA synthetase as well as peroxisomal and mitochondrial β-oxidation were measured in brown adipose tissue homogenates of lean and $\text{ob}$/$\text{ob}$ mice kept at 23°C or acclimated at 4°C. The proton conductance pathway, i.e. the number of purine nucleotide (GDP) binding sites and the percentage of 32,000 polypeptide in brown adipose tissue mitochondria were also measured. In the if$\text{ob}$/$\text{ob}$ mice at 23° C, the specific activities of the palmitoyl CoA synthetase and of the β-oxidation as well as the number of GDB binding sites were lower than in the lean mice by 26%, 43% and 37%, respectively. The percentage of 32, 000 polypeptide, however, was the same in both groups. In the $\text{ob}$/$\text{ob}$ mice at 23° C, the lower homogenate β-oxidation specific activity was due to the fact that the peroxisomal and mitochondrial specific activities were 44% and 37% lower, respectively. Cold acclimation at 4° C was found to cause an increase of the palmitoyl CoA synthetase specific activity, of the palmitoyl CoA synthetase and peroxisomal β-oxidation total activities and of the number of GDP binding sites, in both lean and $\text{ob}$/$\text{ob}$ mice. Cold acclimation increased the percentage of 32,000 polypeptide in the $\text{ob}$/$\text{ob}$ mice only.     

Fluorescence polarisation and composition of membranes in genetic obesity

There was a decrease in the polarisation value of the fluorescent probe diphenylhexatriene in a wide range of purified plasma and subcellular membranes of obese ($\text{ob}\text{ob}$) mice. These changes were consistent with alterations in the fatty acyl chain content of specific membrane phospholipids. An increase in 22:6 and a loss of 18:2 in phosphatidyl ethanolamine was the major compositional change in adipocyte plasma membranes of $\text{ob}\text{ob}$ mice.     

Membrane fluidity and drug metabolism in liver microsomes of lean, obob and dbdb mice

The thermally-induced changes of a cytochrome P-450 dependent activity (ethoxycoumarin-O-deethylase) and of the fluorescence polarization of diphenylhexatriene were compared in microsomes from lean, $\text{ob}\text{ob}$ and $\text{db}\text{db}$ mice. In lean mice, biphasic plots were obtained with break points in the same range of temperature by both methods, whereas, in $\text{ob}\text{ob}$ and $\text{db}\text{db}$ mice, no discontinuities were observed. These results may be related to a modified fatty acid composition of microsomal membranes in mutant mice. They exemplify the influence of the lipid environment on the monooxygenase system as also shown by the modified binding constants of cytochrome P-450 towards type II substrates in $\text{db}\text{db}$ mice.     

Studies on the activity of brown adipose tissue in suckling,pre-obese, obob mice

The properties and activity of brown adipose tissue have been investigated in suckling, pre-obese, $\text{ob}\text{ob}$ mice in order to determine whether decreased thermogenesis in the tissue precedes the development of obesity in this mutant. At 14 days of age there was no difference between the $\text{ob}\text{ob}$ and normal animals in the total amount of interscapular brown adipose tissue, and the DNA content, protein content, and cytochrome oxidase activity of the tissue were similar in the two groups of mice. Respiration rates of brown adipose tissue mitochondria in the presence of albumin were, however, greater in the normal than the $\text{ob}\text{ob}$ animals, although after the addition of GDP to recouple the mitochondria there was no difference between the two groups. The mitochondrial membrane potential, measured with [³H]methyltriphenylphosphonium, was less affected by exogenous GDP in $\text{ob}\text{ob}$ mice than in normal animals. GDP binding to brown adipose tissue mitochondria, an index of the proton conductance pathway, was much greater in normal than in $\text{ob}\text{ob}$ mice at both 10 and 14 days of age; the decreased GDP binding in the mutant animals was found to result from a reduction in the number of binding sites. It is concluded that brown adipose tissue mitochondria of pre-obese $\text{ob}\text{ob}$ mice are more tightly coupled than those of normal siblings, and that the activity of the ‘thermogenic’ proton conductance pathway is lower in the mutant animals. A decrease in thermogenesis in brown adipose tissue is therefore an early event in the development of the $\text{ob}\text{ob}$ mouse and precedes the appearance of obesity.     

A mitochondrial defect in brown adipose tissue of the obese (ob/ob) mouse: reduced binding of purine nucleotides and a failure to respond to cold by an increase in binding.

Atractyloside-insensitive binding of purine nucleotides is reduced in brown adipose tissue mitochondria of the obese ($\text{ob}\text{ob}$) mouse. Exposure of the $\text{ob}\text{ob}$ mouse to 4°C does not induce the usual increase in binding. Atractyloside-insensitive binding of purine nucleotides is believed to be a measure of the heat-producing proton conductance pathway in brown adipose tissue mitochondria. It is, therefore, suggested that the impaired thermogenesis of the $\text{ob}\text{ob}$ mouse is due to a defect in this pathway in the mitochondria of the brown adipose tissue, the major thermogenic tissues in rodents. The greater metabolic efficiency which would result from a reduced operation of this pathway might be the basis for the obesity in the $\text{ob}\text{ob}$ mouse.     

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.     

Characterization of insulin receptor subunits in brain and other tissues by photoaffinity labeling

Specific insulin receptor proteins of plasma membrane preparations from various tissues of the rat were identified using a photoreactive insulin derivative, N^εB29-mono(azidobenzoyl)insulin. Except for the brain, all tissues examined showed the specific photolabeling of two proteins of M_r～130K and ～90K. In brain tissue, only one protein, M_r～115K, was specifically labeled. Liver and adipocyte membranes of the genetic obese ($\text{ob}\text{ob}$) mice showed decreased labeling of both 130K and 90K proteins when compared to those of lean littermates. Labeling of these proteins in liver plasma membranes was abolished by trypsin, whereas neuraminidase increased their electrophoretic mobility in SDS-polyacrylamide gel. The labeling of these two proteins was inhibited by a human anti-receptor serum which also formed an immunocomplex with both proteins. The labeling of the 115K protein in brain tissue was, however, not affected by the antiserum.     

Gender-Specific Metabolomic Profiling of Obesity in Leptin-Deficient ob/ob Mice by 1H NMR Spectroscopy

Despite the numerous metabolic studies on obesity, gender bias in obesity has rarely been investigated. Here, we report the metabolomic analysis of obesity by using leptin-deficient ob/ob mice based on the gender. Metabolomic analyses of urine and serum from ob/ob mice compared with those from C57BL/6J lean mice, based on the ¹H NMR spectroscopy in combination with multivariate statistical analysis, revealed clear metabolic differences between obese and lean mice. We also identified 48 urine and 22 serum metabolites that were statistically significantly altered in obese mice compared to lean controls. These metabolites are involved in amino acid metabolism (leucine, alanine, ariginine, lysine, and methionine), tricarbocylic acid cycle and glucose metabolism (pyruvate, citrate, glycolate, acetoacetate, and acetone), lipid metabolism (cholesterol and carnitine), creatine metabolism (creatine and creatinine), and gut-microbiome-derived metabolism (choline, TMAO, hippurate, p-cresol, isobutyrate, 2-hydroxyisobutyrate, methylamine, and trigonelline). Notably, our metabolomic studies showed distinct gender variations. The obese male mice metabolism was specifically associated with insulin signaling, whereas the obese female mice metabolism was associated with lipid metabolism. Taken together, our study identifies the biomarker signature for obesity in ob/ob mice and provides biochemical insights into the metabolic alteration in obesity based on gender.     

Naltrexone reduces weight gain, alters “β-endorphin”, and reduces insulin output from pancreatic islets of genetically obese mice

Naltrexone, an opiate antagonist, was administered to young obese (ob/ob) and lean mice for five weeks. Animals had continuous access to food and received 10 mg/kg SC twice daily with equivalent volumes of saline given to controls. The effects on body weight, and pituitary and plasma levels of β-endorphin-like material were measured. Naltrexone-injected obese animals gained weight more slowly over the first three weeks while the weight gain of lean animals was not affected by naltrexone. Plasma levels of β-endorphin were shown to be significantly higher in untreated ob/ob mice and this difference increased with age (4–20 weeks). With naltrexone treatment, plasma levels in +/? mice rose and exceeded those in ob/ob. Saline treatment appeared to be a stress, and pituitary β-endorphins rose 4–6 fold in ob/ob compared with +/?. While naltrexone reduced the levels in ob/ob pituitary towards normal, no effect on β-endorphin levels in pituitary of lean mice was obtained. In vitro studies of effects of the opiate antagonists, naloxone, on insulin secretion by isolated islets provided additional evidence of resistance of lean mice to naloxone relative to ob/ob. (IRI secretion fell only in naloxone treated ob/ob islets.) These observations support the contention that this form of genetic obesity is characterized by elevated endogenous opiate levels and an increased sensitivity to opiate antagonists such as naltrexone or naloxone.     

In vivo lipogenesis and enzyme levels in adipose and liver tissues from pair-fed genetically obese and lean rats

Genetically obese Zucker rats pair-fed to lean controls were similar in body weight and food intake, however, epididymal fat pads were considerably larger than lean controls. $\text{In}$$\text{vivo}$ incorporation of acetate-¹⁴C into adipose tissue lipid was not significantly different, however, $\text{in}$$\text{vivo}$ liver lipogenesis was elevated in the obese rat. Characterization of enzyme profiles in both liver and adipose tissues revealed that enzymes normally associated with lipogenesis were elevated in liver tissue from obese rats. Malic enzyme and citrate cleavage enzyme were both depressed in adipose tissue of obese animals. From these data, it appears that the liver may be prominently involved in the development of excessive blood lipid and enlarged fat cells in the Zucker obese rat.     

Naltrexone reduces weight gain, alters “β-endorphin”, and reduces insulin output from pancreatic islets of genetically obese mice

Naltrexone, an opiate antagonist, was administered to young obese (ob/ob) and lean mice for five weeks. Animals had continuous access to food and received 10 mg/kg SC twice daily with equivalent volumes of saline given to controls. The effects on body weight, and pituitary and plasma levels of β-endorphin-like material were measured. Naltrexone-injected obese animals gained weight more slowly over the first three weeks while the weight gain of lean animals was not affected by naltrexone. Plasma levels of β-endorphin were shown to be significantly higher in untreated ob/ob mice and this difference increased with age (4–20 weeks). With naltrexone treatment, plasma levels in +/? mice rose and exceeded those in ob/ob. Saline treatment appeared to be a stress, and pituitary β-endorphins rose 4–6 fold in ob/ob compared with +/?. While naltrexone reduced the levels in ob/ob pituitary towards normal, no effect on β-endorphin levels in pituitary of lean mice was obtained. In vitro studies of effects of the opiate antagonists, naloxone, on insulin secretion by isolated islets provided additional evidence of resistance of lean mice to naloxone relative to ob/ob. (IRI secretion fell only in naloxone treated ob/ob islets.) These observations support the contention that this form of genetic obesity is characterized by elevated endogenous opiate levels and an increased sensitivity to opiate antagonists such as naltrexone or naloxone.     

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.     

Thermogenic and anorectic effects of ephedrine and congeners in mice and rats

(?)-Ephedrine has been shown to increase energy expenditure and cause the loss of body fat in rats and mice. The present study compares the effects of (?)- and (+)-ephedrine, (?)- and (+)- pseudoephedrine, (±)- and (+)-norephedrine and (?)- and (+)-norpseudoephedrine on food intake, energy expenditure and body composition in $\text{ob}$/$\text{ob}$ and normal mice and food intake in rats. The most potent anorectic and thermogenic compounds had the S configuration at the C-2 position but the orders of potencies for the compounds for anorexia and thermogenesis were not identical. The potencies of the compounds in reducing body lipid content correlated better with their thermogenic than their anorectic potencies.     

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.     

Sustained High Protein-tyrosine Phosphatase 1B Activity in the Sperm of Obese Males Impairs the Sperm Acrosome Reaction

Evidence of a causal link between male obesity and subfertility or infertility has been demonstrated previously. However, the mechanism underlying this link is incompletely understood. Here, we report that sustained high protein-tyrosine phosphatase 1B (PTP1B) activity in sperm of obese donors plays an essential role in coupling male obesity and subfertility or infertility. First, PTP1B level and activity were significantly higher in sperm from ob/ob mice than in wild-type littermates. High PTP1B level and activity in sperm was also observed in obese patients compared with non-obese donors. The enhanced sperm PTP1B level and activity in ob/ob mice and obese patients correlated with a defect of the sperm acrosome reaction (AR). Second, treating sperm from male ob/ob mice or obese men with a specific PTP1B inhibitor largely restored the sperm AR. Finally, blockade of sperm AR by enhanced PTP1B activity in male ob/ob mice or obese men was due to prolonged dephosphorylation of N-ethylmaleimide-sensitive factor by PTP1B, leading to the inability to reassemble the trans-SNARE complexes, which is a critical step in sperm acrosomal exocytosis. In summary, our study demonstrates for the first time that a sustained high PTP1B level or activity in the sperm of obese donors causes a defect of sperm AR and that PTP1B is a novel potential therapeutic target for male infertility treatment.     

Liver Glycerol Permeability and Aquaporin-9 Are Dysregulated in a Murine Model of Non-Alcoholic Fatty Liver Disease

One form of liver steatosis, namely Non-Alcoholic Fatty Liver Disease (NAFLD), is a worrisome health problem worldwide characterized by intrahepatic triacylglycerol (TG) overaccumulation. NAFLD is a common feature of metabolic syndrome being often associated with obesity, dyslipidemia and diabetes and mostly closely linked to insulin resistance. The mechanism of NAFLD pathogenesis is object of intense investigation especially regarding complex systems ultimately resulting in excessive TG deposition in hepatocytes. However, scarce is the attention about the relevance of hepatic import of glycerol, the other primary source (as glycerol-3-phosphate) of increased TG in hepatocytes. Obese leptin-deficient (ob/ob) mice, an animal model of NAFLD, were used to evaluate the functional involvement of Aquaporin-9 (AQP9), the major pathway of liver glycerol entry, in hepatosteatosis. By RT-PCR and qPCR, the level of Aqp9 mRNA in the liver of starved obese mice was comparable with the corresponding control lean littermates. By immunoblotting, the AQP9 protein at the hepatocyte sinusoidal plasma membrane of obese mice was markedly lower (33%) than lean mice, a finding fully confirmed by immunohistochemistry. By stopped-flow light scattering, the liver glycerol permeability of ob/ob mice was significantly lower (53%) than lean mice, a finding consistent with both the observed down-regulation of AQP9 protein and increased level of plasma glycerol characterizing obese mice. In summary, our results suggest implication of AQP9 in liver steatosis. The reduction of hepatocyte AQP9 and, consequently, glycerol permeability might be a defensive mechanism to counteract further fat infiltration in liver parenchyma.     

Short‐Term Lenalidomide (Revlimid) Administration Ameliorates Cardiomyocyte Contractile Dysfunction in ob/ob Obese Mice

Lenalidomide is a potent immunomodulatory agent capable of downregulating proinflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and upregulating anti‐inflammatory cytokines. Lenalidomide has been shown to elicit cardiovascular effects, although its impact on cardiac function remains obscure. This study was designed to examine the effect of lenalidomide on cardiac contractile function in ob/ob obese mice. C57BL lean and ob/ob obese mice were given lenalidomide (50 mg/kg/day, p.o.) for 3 days. Body fat composition was assessed by dual‐energy X‐ray absorptiometry. Cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated. Expression of TNF‐α, interleukin‐6 (IL‐6), Fas, Fas ligand (FasL), the short‐chain fatty acid receptor GPR41, the NFκB regulator IκB, endoplasmic reticulum (ER) stress, the apoptotic protein markers Bax, Bcl‐2, caspase‐8, tBid, cytosolic cytochrome C, and caspase‐12; and the stress signaling molecules p38 and extracellular signal‐regulated kinase (ERK) were evaluated by western blot. ob/ob mice displayed elevated serum TNF‐α and IL‐6 levels, fat composition and glucose intolerance, the effects of which except glucose intolerance and fat composition were attenuated by lenalidomide. Cardiomyocytes from ob/ob mice exhibited depressed peak shortening (PS) and maximal velocity of shortening/relengthening, prolonged time‐to‐PS and time‐to‐90% relengthening as well as intracellular Ca²⁺ mishandling, which were ablated by lenalidomide. Western blot analysis revealed elevated levels of TNF‐α, IL‐6, Fas, Bip, Bax, caspase‐8, tBid, cleaved caspase‐3 caspase‐12, cytochrome C, phosphorylation of p38, and ERK in ob/ob mouse hearts, the effects of which with the exception of Bip, Bax, and caspase‐12 were alleviated by lenalidomide. Taken together, these data suggest that lenalidomide is protective against obesity‐induced cardiomyopathy possibly through antagonism of cytokine/Fas‐induced activation of stress signaling and apoptosis.