Role of uncoupling protein 1 in the anti-obesity effect of beta3-adrenergic agonist in the dog.

We have reported that chronic treatment with beta3-adrenoceptor agonists reduces body fat content and induces the expression of mitochondrial thermogenic uncoupling protein 1 (UCP1) in adipose tissue in the dog. To evaluate the role of UCP1 in the anti-obesity effect of the agonists, we isolated adipocytes from subcutaneous fat pad of beagles before and after a 2-week treatment with AJ-9677, a specific beta3-adrenoceptor agonist, and examined their thermogenic activity in vitro. Histological and protein analysis revealed that adipose tissues before the treatment were composed of unilocular cells filled with a single large droplet, while the tissues after the treatment contained many smaller and some multilocular adipocytes expressing UCP1 and abundant mitochondrial proteins. Before the treatment, oxygen consumption rate was very low and did not change even when the cells were stimulated by AJ-9677. Two-week AJ-9677 treatment increased basal oxygen consumption rate by 7-fold, and produced a clear responsiveness to AJ-9677 stimulation. Thus, chronic treatment with AJ-9677 induced UCP1 in adipocytes, where oxygen consumption increased in response to AJ-9677 stimulation. It was suggested that UCP1-dependent energy expenditure in adipose tissue contributes to the anti-obesity effect of beta3-adrenoceptor agonist in dogs.

Beta3-adrenoceptor agonist AJ-9677 reduces body fat in obese beagles.

A selective beta3-adrenoceptor agonist, AJ-9677, was reported to ameliorate obesity and insulin resistance in KK-Ay mice. We examined the acute and chronic effects of AJ-9677 on obese dogs. Oral administration of AJ-9677 (0.01 or 0.1 mg/kg) to overnight fasted obese beagles produced a dose-dependent rise in the plasma levels of non-esterified fatty acids and insulin in 1h, followed by a gradual drop of the plasma glucose level. It produced no apparent abnormal behaviors, but easily detectable cutaneous flushing. Daily treatment of AJ-9677 at a lower dose (0.01 mg/kg) for three weeks produced no notable change in body weight, but at a higher dose (0.1 mg/kg) it reduced the body weight compared to a placebo treatment after seven weeks. Computed tomographic examinations revealed a remarkable reduction of body fat after the AJ treatment, being consistent with the histological observations that the adipose tissue of AJ-9677-treated dogs consisted of smaller and some multilocular adipocytes. The plasma levels of leptin and adiponectin were decreased and increased, respectively, after the AJ treatment, reflecting the reduction of adiposity. It was concluded that AJ-9677 is useful for the treatment of obesity in the dog.

Canine adiponectin: cDNA structure, mRNA expression in adipose tissues and reduced plasma levels in obesity.

Adiponectin is a protein synthesized and secreted by adipocytes. Decreased adiponectin is responsible for insulin resistance and atherosclerosis associated with human obesity. We obtained a cDNA clone corresponding to canine adiponectin, whose nucleotide and deduced amino acid sequences were highly identical to those of other species. Adiponectin mRNA was detected in adipose tissues, but not in other tissues, of dogs. When 22 adult beagles were given a high-energy diet for 14 weeks, they became obese, showing heavier body weights, higher plasma leptin concentrations, but lower plasma adiponectin concentrations. The adiponectin concentrations of plasma samples collected from 71 dogs visiting veterinary practices were negatively correlated to plasma leptin concentrations, being lower in obese than non-obese dogs. These results are compatible with those reported in other species, and suggest that adiponectin is an index of adiposity and a target molecule for studies on diseases associated with obesity in dogs.

Synthesis and Antifungal Activity of Some Organotin(IV) Carboxylates.

Six diorganotin(IV) carboxylates prepared by reacting diorganotin(IV) dichlorides with the respective silver carboxylate have been tested for antifungal activity against Aspergillus. niger, Aspergilluus flavus and Pencillium. citrinum in Sabourand dextrose broth. The compounds generally exhibit greater fungitoxicity than the diorganotin(IV) dichlorides and the carboxylic acids from which they were synthesized. In keeping with the generally accepted notion that the organotin moiety plays an important role in deciding the antifungal activity of an organotin compound, the diphenyltin(IV) compounds were more active than their di-n-butyltin(IV) analogues. However, the order of increasing fungitoxicity of the compounds parallels that of the uncomplexed carboxylic acids. The implications of the results are discussed.

Changes in thyroid state affect pHi and Nai+ homeostasis in rat ventricular myocytes.

We have tested the hypothesis that thyroid state may influence both the flow of cellular Ca2+ and the myofilament response to Ca2+ by effects on intracellular pH (pHi) and Na+ (Nai+). Single cardiac myocytes isolated from hypothyroid, euthyroid and hyperthyroid animals were loaded with fura-2/AM (Cai2+ probe), BCECF/AM (pHi probe) or SBFI/AM (Nai+ probe). Compared with hypothyroid animals, myocytes isolated from hyperthyroid rat hearts demonstrated a significant: (1) increase in extent of shortening; (2) decrease in the time to peak contraction; (3) increase in the peak amplitude of the fura-2 fluorescence ratio; (4) decrease in pHi (DeltapHi=0. 19+/-0.05); and (5) increase in Nai+ (DeltaNai+=2.88+/-0.55 mM). We have also compared pHi in Langendorff perfused hypo- and hyperthyroid rat hearts using NMR. We have found that hyperthyroid hearts are 0.15+/-0.03 pH units more acidic than hypothyroid hearts. Analysis of mRNA levels demonstrated that hyperthyroidism increased expression of both the Na+/Ca2+ exchanger and Na+/H+ antiporter, and decreased expression of Na+ channel mRNAs. These changes appear partially responsible for the observed changes in Nai+ and pHi. Our results provide the first evidence that changes in cardiac contractility associated with altered thyroid state not only involve effects on Ca2+, but may also involve changes in the response of the myofilaments to Cai2+mediated by altered pHi and Nai+.

Inhibition of the calcium paradox in isolated rat hearts by high perfusate sucrose concentrations.

If a colloid osmotic (oncotic) pressure gradient develops across the myocyte membrane during the calcium paradox, adding an oncotic agent to the perfusate should be inhibitory. After 10-min perfusion with Ca(2+)-free Krebs-Henseleit (KH) buffer under constant flow at 34 degrees C, myoglobin release was measured from Langendorff hearts reperfused with Ca(2+)-containing KH buffer. When the Ca(2+)-free medium contained 200 mM sucrose, myoglobin release was reduced to 5% of that observed in the absence of sucrose, a change that was not seen when 200 mosM NaCl, choline chloride, LiCl, or glycerol was added. Replacement of 75 mM NaCl in the perfusate with 150 mM sucrose resulted in myoglobin release values that were 4% of the control. Plots of myoglobin release against sucrose concentration under these hypertonic and isotonic conditions yielded similar though separate curves. Sucrose also inhibited increases in wet weight-to-dry weight ratio and decreases in ATP and phosphocreatine contents. These results support the hypothesis that an oncotic pressure gradient arises during the calcium paradox at the moment of increased membrane permeability and plays a major role in its development.

Inhibition of chloride binding to the anion transport site by diethylpyrocarbonate modification of Band 3.

The line widths of 35Cl- nuclear magnetic resonances were used to measure chloride binding by Band 3. Since this procedure relates directly to binding, the data obtained may be interpreted more unequivocally than affinities derived from kinetic data which could be related to either translocation or binding. Chloride binding to the active sites in Band 3 was assessed from that portion of the total line width which was sensitive to 4,4'-dinitrostilbene-2,2'-disulfonic acid. These sites appeared to be completely inhibited by treatment of erythrocyte membranes with diethylpyrocarbonate. This result is consistent with our previous observation that this reagent inhibits anion transport in resealed erythrocyte ghosts (Izuhara, Okubo & Hamasaki, 1989, Biochemistry 28:4725-4728). Hydroxylamine could not reverse the diethylpyrocarbonate inhibition of chloride binding to Band 3. The pH-dependence of diethylpyrocarbonate reactivity suggests that the modified residues may be those of histidine.

The pH dependence of red cell membrane transport of titratable anions studied by NMR spectroscopy.

The effects of varying extracellular pH on the rates of uptake of titratable anions by human erythrocytes under conditions of constant intracellular pH have been determined for a series of highly related anions, the phosphate "analogs." These compounds are simply substituted phosphorus oxyacids, differing in the number and acidity of titratable protons: phosphate (HPO4(2-), pKa 6.8); phosphite (HPO3(2-), pKa 6.4); hypophosphite (H2PO2-); methylphosphonate ((CH3)PO3(2-), pKa 7.4); dimethylphosphinate ((CH3)2PO2-); fluorophosphate [PO3F2-, pKa 4.7); and thiophosphate (HSPO3(2-), pKa 5.5). Suspensions of intact, Cl(-)-loaded erythrocytes (intracellular pH, 7.2) were incubated at 37 degrees C in isotonic buffers (pH 4-8) containing 60 mM phosphate analog for specified time intervals, whereupon influx was halted by the addition of 1 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), an inhibitor of anion exchange. The intracellular anion concentrations were determined from 31P or 19F nuclear magnetic resonance spectra from the erythrocyte suspensions. The influx rates for the titratable phosphate analogs exhibited bimodal pH dependence, reaching maximal levels at pH values that increased with increasing anion pK. This pH-dependent behavior is consistent with a transport channel that contains a titratable regulatory site which interacts with the translocated anion. Based upon the Henderson-Hasselbalch equation, the probability that a titratable anion will have an electric charge of equal magnitude to that of the titratable carrier is highest at a pH value exactly midway between the pK of the regulatory site and that of the anion. The pH maxima observed for the phosphate analogs indicate a pK for this site of 5.5 at 37 degrees C. Intracellular pH changes associated with influx indicated that transport of the "fast" anion phosphite is largely in monoionized form. Intracellular pH changes associated with transport of slow anions were predominantly determined by partial ionic equilibrium effects and did not indicate the ionization state of the transported anion.

Erythrocyte anion transport of phosphate analogs.

The phosphate analogs are a series of chemically related small anions based upon tetrahedrally bonded phosphorus. Each compound is a mono- or disubstituted phosphorus oxyacid. These chemical substitutions lead to differences in the number and acidity of titratable protons, differences in molecular structures and charge distributions, and unique 31P, 19F, or 1H nuclear magnetic resonance spectra for each analog. These compounds include phosphate, phosphite, hypophosphite, fluorophosphate, thiophosphate, methylphosphonate, and dimethylphosphinate. NMR spectra were obtained from human erythrocytes suspended in buffers containing phosphate analogs. Intracellular and extracellular 31P and 19F chemical shifts of these anions were found to be nonequivalent, due to magnetic susceptibility differences between the two compartments, as well as to the transmembrane pH gradient. NMR spectroscopy was used to measure erythrocyte influx rates of the phosphate analogs, as well as the intracellular and extracellular pH changes that accompany influx, in red cell suspensions incubated for selected time intervals. Anion influx rates were found to vary over three orders of magnitude among the phosphate analogs. All analogs showed concentration-dependent influx rate saturation. The major determinant of influx rates was neither the molecular weight of the analog nor the net charge on the anion, but rather the structure of the anion. Phosphite (HPO2-3), the anion most closely resembling bicarbonate (a natural substrate for anion exchange) was found to have the highest influx rate.

The pH dependence of red cell membrane transport of titratible anions. An NMR study.

The titratible carrier model for erythrocyte anion transport predicts that the pH dependence of titratible anion transport should differ considerably from that for monovalent or divalent anions. The effects of pH on titratible anion influx rates have been determined for a series of related anions, the phosphate "analogs". These compounds are simply substituted phosphorus oxyacids, differing in the number and acidity of titratible protons. Influx of each analog at 60 mM concentration into Cl- loaded erythrocytes at constant intracellular pH (7.2) was measured over an extracellular pH range of 4.0 to 8.0 using 31P or 19F nuclear magnetic resonance spectroscopy. Influx rates peaked at pH values that increased with increasing anion pK. Using the Henderson-Hasselbalch equation to determine the effects of pH on the charges carried by a titratible anion and a titratible carrier, the pH at which the anion would most likely have the same charge as the carrier was found to be exactly midway between their respective pK values. The observed pH maxima for the phosphate analog influx curves are consistent with transport by a titratible carrier having a pK of 5.5.

A Bartter's-like syndrome from capreomycin, and a similar gentamicin tubulopathy.

Marked renal potassium and magnesium wasting, alkalosis, and a progressive increase in plasma renin and eventual hyperaldosteronemia developed during a 15-month course of in-hospital capreomycin therapy that was necessary for drug-resistant pulmonary tuberculosis. A prominent feature of the present case was renal chloride wasting, a feature of the capreomycin syndrome that has previously received little attention. Similar potentially life-threatening metabolic abnormalities, which resemble those found in Bartter's syndrome, can occur during prolonged therapy with the antibiotic gentamicin. In the present case, electrolyte abnormalities were unaffected by three days of indomethacin therapy but were partially corrected by large doses of spironolactone. Capreomycin, viomycin (an antibiotic closely related to capreomycin), and gentamicin are highly basic polypeptide antibiotics that may induce strikingly similar and potentially fatal syndromes of renal tubular dysfunction that can feature multiple electrolyte abnormalities.

Elimination of the settling artifact in the nuclear magnetic resonance spectroscopy of living cells.

Nuclear magnetic resonance spectroscopy has become a powerful tool for metabolic investigations on living cell suspensions. However, unless mechanical means are used to maintain the cells in dispersion, settling occurs during the NMR experiment. Because high packed-cell volumes are generally used to produce maximum NMR signals, settling may be inapparent to the eye, leading to unrecognized artifactual changes in NMR spectra. Such artifacts include time-dependent loss of signal intensity when the sample volume approximates the sensitive volume of the NMR probe, and time-dependent increase in signal intensity when the sample volume exceeds the sensitive volume. Through the addition of the polysaccharide arabinogalactan, increasing the buoyant density of the suspending medium to approach that of the cells, we have eliminated cell settling and improved the quality of 31P NMR spectra of human erythrocytes.

Erythrocyte membrane lysophospholipase activity in muscular dystrophy.

The membrane lysophospholipase activity of erythrocytes obtained from Duchenne muscular dystrophy patients was lower than that of erythrocytes from age-matched normal boys. On the other hand, the membrane enzyme activity of erythrocytes from myotonic dystrophy patients was not different from that of their age- and sex-matched controls. Dipyridamole (0.1 mM) and glycerol 3-phosphorylcholine (2 mM) had no significant effect on these enzyme activities. These results suggest that membrane lysophospholipid metabolism may be altered in Duchenne muscular dystrophy but not in myotonic dystrophy.

Lysophosphatidylcholine-induced lysis of erythrocytes in Duchenne and myotonic dystrophies and in Huntington's disease.

Erythrocytes from Duchenne dystrophy patients lysed more readily than red cells from age-matched normal boys when lysophosphatidylcholine (LPC) concentrations that caused 50% lysis were compared. Erythrocytes from myotonic dystrophy patients appeared to be more resistant than cells from age-matched normal adults at certain medium LPC concentrations. Erythrocytes from patients with Huntington's disease showed no significant differences from erythrocytes of normal adults. Thus, the manner in which erythrocytes respond to LPC may reflect the putative membrane alterations in these diseases. Inhibition of LPC-induced lysis by 0.1 mM dipyridamole was observed in all groups. Since this agent did not inhibit LPC lysis at 0 degrees C, its action at 37 degrees C could be related to activation of a membrane enzyme. On the other hand, dipyridamole decreased osmotic fragility at 0 degrees C and 37 degrees C indicating that a physical change in membrane structure may be the primary alteration produced by this agent.

Erythrocyte phosphate content in Huntington's disease.

Total phosphate was higher in erythrocytes of patients with Huntington's disease (HD) than in erythrocytes of age- and sex-matched normal adults. A more rapid rate of organic phosphate degradation was also noted in HD erythrocytes during metabolic depletion. These variations may be associated with alterations of membrane permeabilities of HD erythrocytes to inorganic phosphate and to cations.

Studies on (K+ + H+)-ATPase. IV. Effects of phospholipase C treatment.

(1) The total phospholipid content of a gradient purified (K+ + H+)-ATPase preparation from pig gastric mucosa is 105 mumol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 mumol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The (K+ + H+)-ATPase and K+ stimulated p-nitrophenylphosphatase activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, Km value for ATP and temperature dependence of the gastric (K+ + H+)-ATPase, but slightly decreases the Ka value for K+. (5) Phospholipase C treatment lowers the AdoPP[NH]P binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules.

Erythrocytes in muscular dystrophy. Investigation with 31P nuclear magnetic resonance spectroscopy.

Phosphorus 31 nuclear magnetic resonance (31P NMR) signals were recorded from intact human erythrocytes for 16 hours. Total phosphate concentration, which was estimated as the sum of the individual 31P signals, was 25% lower in erythrocytes from men with myotonic dystrophy than in control erythrocytes. The inorganic-phosphate fraction contained the highest average phosphate concentration over the 16-hour period, and made the major contribution to the difference in total phosphate between the two groups. This result was not observed in erythrocytes from either women with myotonic dystrophy or patients with Duchenne's dystrophy and may be due to a change in cell membrane permeability to inorganic phosphate, which leads to lower steady-state concentrations of the intracellular phosphates.

Erythrocyte metabolism in muscular dystrophy.

Erythrocytes from patients with Duchenne's and myotonic muscular dystrophies contained more adenosine triphosphate (ATP) and produced more lactate than did normal erythrocytes in incubation studies conducted in vitro at an initial pH of 7.4. Since the same results were obtained in two different genetic dystrophies, these metabolic variations appear to be secondary to the primary changes occurring in these diseases. Following ouabain treatment, ATP content increased and lactate production decreased in erythrocytes from both dystrophies. This result differs from one reported earlier in experiments conducted at alkaline pH.