Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase.

Adiponectin (Ad) is a hormone secreted by adipocytes that regulates energy homeostasis and glucose and lipid metabolism. However, the signaling pathways that mediate the metabolic effects of Ad remain poorly identified. Here we show that phosphorylation and activation of the 5'-AMP-activated protein kinase (AMPK) are stimulated with globular and full-length Ad in skeletal muscle and only with full-length Ad in the liver. In parallel with its activation of AMPK, Ad stimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acid oxidation, glucose uptake and lactate production in myocytes, phosphorylation of ACC and reduction of molecules involved in gluconeogenesis in the liver, and reduction of glucose levels in vivo. Blocking AMPK activation by dominant-negative mutant inhibits each of these effects, indicating that stimulation of glucose utilization and fatty-acid oxidation by Ad occurs through activation of AMPK. Our data may provide a novel paradigm that an adipocyte-derived antidiabetic hormone, Ad, activates AMPK, thereby directly regulating glucose metabolism and insulin sensitivity in vitro and in vivo.

The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity.

Adiponectin is an adipocyte-derived hormone. Recent genome-wide scans have mapped a susceptibility locus for type 2 diabetes and metabolic syndrome to chromosome 3q27, where the gene encoding adiponectin is located. Here we show that decreased expression of adiponectin correlates with insulin resistance in mouse models of altered insulin sensitivity. Adiponectin decreases insulin resistance by decreasing triglyceride content in muscle and liver in obese mice. This effect results from increased expression of molecules involved in both fatty-acid combustion and energy dissipation in muscle. Moreover, insulin resistance in lipoatrophic mice was completely reversed by the combination of physiological doses of adiponectin and leptin, but only partially by either adiponectin or leptin alone. We conclude that decreased adiponectin is implicated in the development of insulin resistance in mouse models of both obesity and lipoatrophy. These data also indicate that the replenishment of adiponectin might provide a novel treatment modality for insulin resistance and type 2 diabetes.

Inhibition of RXR and PPARgamma ameliorates diet-induced obesity and type 2 diabetes.

PPARgamma is a ligand-activated transcription factor and functions as a heterodimer with a retinoid X receptor (RXR). Supraphysiological activation of PPARgamma by thiazolidinediones can reduce insulin resistance and hyperglycemia in type 2 diabetes, but these drugs can also cause weight gain. Quite unexpectedly, a moderate reduction of PPARgamma activity observed in heterozygous PPARgamma-deficient mice or the Pro12Ala polymorphism in human PPARgamma, has been shown to prevent insulin resistance and obesity induced by a high-fat diet. In this study, we investigated whether functional antagonism toward PPARgamma/RXR could be used to treat obesity and type 2 diabetes. We show herein that an RXR antagonist and a PPARgamma antagonist decrease triglyceride (TG) content in white adipose tissue, skeletal muscle, and liver. These inhibitors potentiated leptin's effects and increased fatty acid combustion and energy dissipation, thereby ameliorating HF diet-induced obesity and insulin resistance. Paradoxically, treatment of heterozygous PPARgamma-deficient mice with an RXR antagonist or a PPARgamma antagonist depletes white adipose tissue and markedly decreases leptin levels and energy dissipation, which increases TG content in skeletal muscle and the liver, thereby leading to the re-emergence of insulin resistance. Our data suggested that appropriate functional antagonism of PPARgamma/RXR may be a logical approach to protection against obesity and related diseases such as type 2 diabetes.

Gangliosides expressed in human breast cancer.

Breast tumors that were histopathologically diagnosed as invasive ductal carcinoma were examined in relation to their abnormal expression of gangliosides. Total ganglioside levels that were expressed as lipid-bound sialic acids were significantly higher in breast tumor tissues than in normal mammary tissues. Two kinds of unusual gangliosides were found to be expressed in many cases of breast tumors. One was a group of O-acetylated gangliosides, such as O-acetyl-GD3 and O-acetyl-GT3. They are known as fetal gangliosides, which appear in fetal brains. The other was an N-glycolylneuraminic acid-containing ganglioside, N-glycolyl-GM3, which had not been previously found in normal human tissues. The finding that unusual gangliosides are expressed in breast tumors may provide the basis for their immunological diagnosis and vaccine therapy.

Altered neurotrophic factors' expression profiles in the nucleus of the solitary tract of spontaneously hypertensive rats.

AIM: Our previous findings suggest that the nucleus of the solitary tract (NTS), a pivotal region for regulating the set point of arterial pressure, exhibits abnormal inflammation in pre-hypertensive and spontaneously hypertensive rats (SHRs), with elevated anti-apoptotic and low apoptotic factor levels compared with that of normotensive Wistar-Kyoto (WKY) rats. Whether this chronic condition affects neuronal growth and plasticity in the NTS remains unknown. To unveil the characteristics of the neurodevelopmental environment in the NTS of SHRs, we investigated the expression of neurotrophic factors transcripts in SHRs. METHODS: RT(2) Profiler PCR Array targeting rat neurotrophins and their receptors was used to screen for differentially expressed transcripts in the NTS of SHRs compared to that of WKY rats. Protein expression and physiological functions of some of the differentially expressed transcripts were also studied. RESULTS: Gene and protein expressions of glial cell line-derived neurotrophic factor family receptor alpha-3 (Gfrα-3) factor were both upregulated in the NTS of adult SHRs. Gene expressions of corticotropin-releasing hormone-binding protein (Crhbp), interleukin-10 receptor alpha (Il-10ra) and hypocretin (Hcrt) were downregulated in the NTS of adult SHRs. The Gfrα-3 transcript was increased and the Hcrt transcript was decreased in the NTS of young pre-hypertensive SHRs, suggesting that these profiles are not secondary to hypertension. Moreover, microinjection in the NTS of hypocretin-1 decreased blood pressure in adult SHRs. CONCLUSION: These results suggest that altered neurotrophic factors transcript profiles may affect the normal development and function of neuronal circuitry that regulates cardiovascular autonomic activity, thereby resulting in manifestations of neurogenic hypertension in SHRs.

A novel ganglioside, 9-O-acetyl GD1b, is recognized by serum antibodies in Guillain-Barré syndrome.

A hitherto undescribed ganglioside was detected in a crude ganglioside fraction of bovine brain using an IgM M-protein binding to Gal beta 1, 3GalNAc residue. We purified and identified it as 9-O-acetyl GD1b based on results of alkali treatment that yielded GD1b and results of fast atom bombardment-mass and gas chromatography-mass spectrometries. 9-O-acetyl GD1b was also found to be present in human peripheral nerve tissue. The reactivities of the serum antibodies from patients with Guillain-Barré syndrome to 9-O-acetyl GD1b, GD1b, and GM1 were determined by ELISA and TLC immunostaining. Nineteen of 85 serum samples from Guillain-Barré syndrome patients had antibodies that bound to 9-O-acetyl GD1b: 14 of the positive samples also reacted with GM1 and GD1b, three reacted with GM1 but not with GD1b, one with GD1b but not with GM1, and one with neither GM1 nor GD1b. These results show that a subset of patients with Guillain-Barré syndrome had antibodies that react with 9-O-acetyl GD1b; therefore, this ganglioside can serve as a target antigen against the antibodies present in Guillain-Barré syndrome.

Spaceflight alters the fiber composition of the aortic nerve in the developing rat.

Hydrostatic pressure gradients due to the gravitational force in blood vessels disappear under conditions of microgravity during spaceflight, and the ability of the baroreceptor reflex to control arterial pressure and blood distribution may be altered. We hypothesized, on the basis of the results obtained in our previous experiments using the head-down tilt method in rats and rabbits, that the range of increase in arterial pressure caused by animal behavior narrows under conditions of microgravity, affecting the development of high-threshold unmyelinated fibers in the rat aortic nerve which sends signals from baroreceptors located in the aortic wall to the reflex center. We verified this hypothesis using 9-day-old rat neonates housed with their dams for 16 days on the space shuttle Columbia in outer space (STS-90, Neurolab Mission). Age-matched neonatal rats with the dams remained on the ground as controls. After breeding was carried out in the three experimental groups (FLT, spaceflight; AGC, asynchronous ground control; VIV, vivarium ground control), specimens of the 25-day-old rats were excised and five left aortic nerves in each group were examined by electron microscopy. The number of aortic unmyelinated fibers was significantly less in the FLT group than in each ground control (mean+/-S.D.; 139+/-37 in the FLT, 207+/-36 in the AGC, 283+/-121 in the VIV; P<0.05), which may be related to the weakness of the baroreceptor reflex under conditions of microgravity in space. This result may contribute to understanding of the several cardiovascular issues which occur under microgravity and after reexposure to gravity in human.

A new O-acetylated trisialoganglioside, 9-O-acetyl GT2, in cod brain.

An O-acetylated trisialoganglioside that is converted to GT2 on mild base treatment was found in cod brain. This alkali-labile ganglioside was isolated using high-performance liquid chromatography, and its chemical structure was characterized. This novel ganglioside was identified as a GT2 derivative having an acetyl group at the C-9 position of the external sialic acid. Its chemical structure is as follows.

Isolation and characterization of a trisialyllactosylceramide, GT3, containing an O-acetylated sialic acid in cod fish brain.

An O-acetylated ganglioside that generated a trisialyllactosylceramide or GT3 by base treatment was found for the first time in cod fish brain. This ganglioside was isolated by high-performance liquid chromatography, and characterized by fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy in addition to chemical analysis. The structure was identified as a modified GT3 in which the external sialic acid is O-acetylated at the C-9 position. The chemical structure is as follows: II3(9-O-Ac-NeuAc2-8NeuAc2-8NeuAc2-3)Lac Cer.

Characterization of a novel triphosphonooctaosylceramide from the eggs of the sea hare, Aplysia kurodai.

We have reported the existence of a triphosphonoglycosphingolipid, EGL-I, in the eggs of a sea gastropod, Aplysia kurodai [Yamada, S., Araki, S., Abe, S., Kon, K., Ando, S., and Satake, M. (1995) J. Biochem. 117, 794-799]. We have now isolated a novel glycosphingolipid, named EGL-II, from the eggs of Aplysia. By component analysis, sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, secondary ion mass spectrometry, and proton magnetic resonance spectrometry, its structure was revealed to be as follows: Galalpha1-->3(GlcNAcalpha1-->2)Galalpha1-->3(3-O-MeGalalpha1-->2)Galalpha1-->3[6'-O-(2-aminoethylphosphonyl)Galalpha1-->2](2-aminoethylphosphonyl-->6)Galbeta1-->4(2-aminoethylphosphonyl-->6)Glcbeta1-->1ceramide. The major aliphatic components of the ceramide are palmitic acid, stearic acid, and anteisononadeca-4-sphingenine.

Gangliosides and sialylcholesterol as modulators of synaptic functions.

Gangliosides were shown to enhance the release of acetylcholine from synaptosomes on stimulation. The influx of calcium ion into synaptosomes on membrane depolarization was increased by gangliosides. This was hypothesized to be an underlying mechanisms for the enhancement of acetylcholine release. Studies using calcium channel blockers revealed that four distinct types of voltage-dependent calcium channels occurred in cerebrocortical synapses, and that the N-type was primarily responsible for the evoked release of acetylcholine. An additional result suggests that gangliosides may act mainly on the N-type calcium channel. Cholinergic-specific gangliosides, Chol-1 alpha, were assumed to participate in the mechanism of high-affinity choline uptake. These two different actions of gangliosides were found to be mimicked by synthetic ganglioside analogs. Calcium influx was increased by alpha-sialylcholesterol, and choline uptake was accelerated by beta-sialylcholesterol. Gangliosides and sialylcholesterol having these apparently beneficial effects were shown to ameliorate decreased functions of synapses from aged brains.

Gangliosides enhance KCl-induced Ca2+ influx and acetylcholine release in brain synaptosomes.

Effects of gangliosides GM1 and GQ1b on cholinergic synaptic functions were investigated using synaptosomes prepared from mouse brain cortices. Treatment of synaptosomes with GM1 and GQ1b increased high K(+)-evoked acetylcholine (ACh) release in a bell-shaped dose-dependent manner. The peaks of the effects were found to be at 1-5 microM for GM1 and 5-10 microM for GQ1b. ACh synthesis and the levels of ACh in synaptosomes were not affected by the ganglioside treatment. Both gangliosides enhanced depolarization-induced influx of calcium ions into synaptosomes. These results indicate that GM1 and GQ1b gangliosides increase evoked ACh release by modulating voltage-dependent calcium channels in the synaptic plasma membranes. The effect of GM1 on calcium ion influx remained after repetitive washings, but was almost completely abolished when the bound GM1 was removed by trypsin. This indicates that the fraction of GM1 which was tightly bound to, but not incorporated in synaptic plasma membranes, is responsible for activating the calcium channels.

Characteristics of gangliosides including O-acetylated species in growth cone membranes at several developmental stages in rat forebrain.

Growth cones, the motile tips of extending neuronal processes, are involved in accurate synaptogenesis. To study the developmental changes in ganglioside composition including O-acetylated gangliosides in growth cones, we analyzed the gangliosides in growth cone membranes (GCM) prepared from rat forebrains at different developmental stages. At several stages, GCM contained significantly larger amounts of gangliosides than the other membrane subfractions. The ganglioside content of GCM increased in amount with development. Moreover, in GCM, the relative amount of GD3 gradually decreased, and that of GD1a dramatically increased. There were significant differences in the composition of ganglioside species between GCM and the perinuclear plasma membrane subfraction (NM); most importantly, GCM had a higher ratio of GD1a to GM3 plus GD3 than NM. There were three different O-acetylated gangliosides in GCM: O-acetyl-GD3, O-acetyl-GT1b, and O-acetyl-GQ1b. The molar ratio of O-acetyl-GD3 decreased in GCM at later stages (5% of the total gangliosides at embryonic day 17, to 1% at postnatal day 5). However, those of the other two O-acetylated gangliosides were almost constant (1-2% of the total). Our results show that there are significant differences in ganglioside content and composition between the membrane subfraction of growth cones and the perinuclear portion. This suggests that several species of gangliosides, including O-acetyl-GD3, play a role in growth cone function.

Differences in lipid composition between isolated growth cones from the forebrain and those from the brainstem in the fetal rat.

The lipid composition of nerve growth cone membranes isolated from rat fetal forebrain or brainstem by the sucrose density gradient method was analyzed biochemically and immunochemically. In the forebrain, growth cone membrane (GCM) contained lower levels of gangliosides than those from other heavier fractions, but it was not the case in the fetal brainstem at the same developmental stage. The distinctive features in the ganglioside composition of GCM are the predominance of GD3 and the presence of c-series gangliosides that are due to fetal expression in mammals. A unique acidic glycolipid, sulfoglucuronylparagloboside (SGPG), which is not present in adult brains, was first detected in both forebrain and brainstem GCM. Including such minor species, the ganglioside composition in forebrain or brainstem GCM was almost identical to other membrane fractions from the forebrain or brainstem. The compositional ratios of the major lipid classes in membranes, cholesterol and phospholipids, seemed to be common to forebrain GCM and brainstem GCM, as indicated by the identical values of phospholipid-to-protein (PL/Pr), cholesterol-to-protein (Ch/Pr), and cholesterol-to-phospholipid (Ch/PL) ratios for both. This study has revealed that GCM isolated from forebrain which is supposed to be at an earlier stage of neuronal differentiation than brainstem has less amounts of total gangliosides, high proportion of GD3 to GD1a and enriched c-series gangliosides as compared to brainstem GCM.

Differential fatty acid release from CA1 and CA3 regions of rat hippocampal slices under hypoxia and hypoglycemia.

Rat hippocampal slices were subjected to hypoxia and/or hypoglycemia for 10 min, and free fatty acids released in CA1 and CA3 regions were separately analyzed. Fatty acid accumulation in CA1 was not so significant under hypoglycemia, but very prominent under hypoxia. Free fatty acid levels in CA3 were much less than those in CA1 even under hypoxia plus hypoglycemia. This observation seems to be consistent with the selective vulnerability of CA1 neurons seen in in vivo ischemia. The decreasing order of accumulation of free fatty acid species in CA1 was C16:0 > C18:0 > C18:1 > C20:4 > C22:6. The increment fold as compared to control level was decreasing as follows: C22:6, 28 times; C20:4, 13 times, C18:1, 10 times; C18:0 = C16:0, 3 times. The present experimental conditions using hippocampal slices provided a good in vitro model to prove the selective hypoxic damages of the CA1 subfield in terms of free fatty acid release in association with the membrane degradation.

Age-related changes in the uptake of calcium channel blockers by brain capillary endothelial cells and synaptosomal fractions.

The effects of aging on the uptake of calcium channel blockers through brain capillaries to brain parenchyma were investigated. Two different piperazine derivatives, flunarizine and 1-[bis(fluorophenyl)-methyl]-4-(2,3,4-trimethoxybenzyl)piperazine (abbreviated as KB-2796), were orally administered to young and aged rats. The concentrations of the blockers in plasma, endothelial cells of the brain capillaries and synaptosomal fractions of the three brain regions were determined by high-performance liquid chromatography. Flunarizine was more incorporated into brain tissues than KB-2796 in both young and aged rats. The uptake efficiency of KB-2796 from the circulation to the brain decreased more remarkably than that of flunarizine in aged rats. The partition coefficients between n-octanol and water indicated that KB-2796 was less hydrophobic than flunarizine. These results suggest that the uptake of the chemicals seems to be influenced by their hydrophobicity and the age of animals given.

Enhancement of sensitivity of ion-exchanger absorptiometry by using a thick ion-exchanger layer.

Enhancement of the sensitivity of ion-exchanger absorptiometry by the use of much thicker ion-exchanger layers than those previously employed has been investigated. Because the background attenuance due to light-scattering from the solid particle layer increases only moderately, whereas the net absorbance of the sorbed sample species increases greatly when the cell length is increased, a cell with at least 10-mm path-length may be effectively used without difficulty in most commercial spectrophotometers, giving at least ten times the sensitivity obtainable with a 1-mm cell. Since the background attenuance depends on the optical geometry, it can be lowered to some extent by using a particular type of spectrophotometer, and by placing a cylindrical mirror between the cell and the light-detector window or by setting the cell as close as possible to the window. The use of such long cells has been found to improve the sensitivity in the chromium-diphenylcarbazide, iron-1,10-phenanthroline, nickel-PAN, cadmium-PAR, bismuth-chloride and uranium-thiocyanate systems. Other methods may be similarly improved.

Ion-exchanger ultraviolet spectrophotometry for uranium(VI).

A sensitive method based on solid-phase spectrophotometry has been developed for the microdetermination of uranium(VI) in water samples. Uranium is sorbed on the anion-exchanger QAE-Sephadex from thiocyanate solution and the absorbance of the exchanger is measured at 300 nm. This method is about 30 times more sensitive than solution spectrophotometry. Absorption spectra of various metals in the anion-exchanger phase are presented and their interferences discussed. A procedure for the cation-exchange separation of uranium from accompanying elements before spectral measurement of uranium is proposed.

Some investigations on new polyphosphates by the anion-exchange method.

The structure of the pentamer (a new P-P bonded phosphate) produced by the hydrolysis of dodecaoxohexaphosphoric(III) acid (ring hexamer) has been examined by an anion-exchange distribution method after complex formation with copper(II) and found to be HPO(OH)PO(OH)PO(OH)PO(OH)PO(OH)H. The hexamer and pentamer salts of these P-P bonded linear phosphorus anions have been prepared. The yield of each species was about 1 mole% of the parent material.

Ion-exchanger phase absorptiometry for trace analysis.

Ion-exchanger phase absorptiometry is based on the direct measurement of the degree of light-absorption by an ion-exchange resin phase which has sorbed a sample component. Direct application of the method makes it possible to determine trace elements in natural water samples without preconcentration. In this paper, all systems hitherto developed are reviewed and the theoretical background of solid-phase absorptiometry is described.