Biomedical subjects

H A Lardy

Publications and source records attributed to H A Lardy.

At least 55 records · Page 3Linked to original sources

PubMed1987

Carnitine supplementation and depletion: tissue carnitines and enzymes in fatty acid oxidation.

Sixty-two male rats were randomly assigned into a 3 X 2 X 2 factorial design containing 12 groups according to carnitine treatment, exercise training (treadmill, 1 h, 5 times/wk, 8 wk, 26.8 m/min, 15% grade), and physical activity [rested for 60 h before they were killed or with an acute bout of exercise (1 h, 26.8 m/min, 15% grade) immediately before they were killed]. Isotonic saline was injected intraperitoneally 5 times/wk in the controls, whereas 750 mg/kg of L- or D-carnitine, respectively, were injected in the supplemented and depleted treatment groups. A significant increase in free and short-chain acyl carnitine concentration in skeletal muscle and heart was observed in L-carnitine supplemented rats, whereas a significant reduction in skeletal muscle, heart, and liver occurred in rats depleted of L-carnitine. Long-chain acyl carnitine in all tissues was not altered by carnitine treatment; training increased plasma and liver concentrations, whereas acute exercise decreased skeletal muscle and increased liver concentrations. An acute bout of exercise significantly increased short-chain acylcarnitine in liver, regardless of carnitine and/or training effects. beta-Hydroxyacyl-CoA dehydrogenase activity in skeletal muscle was induced by training but reduced by depletion. Carnitine acetyltransferase (CAT) was significantly increased in heart by L-carnitine supplementation, whereas it was reduced by depletion in skeletal muscle. Exercise training significantly increased CAT activity in skeletal muscle but not in heart, whereas acute exercise significantly increased activity in both tissues. Carnitine palmitoyltransferase activity was increased by acute exercise in the heart in only the supplemented and exercise-trained rats.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1987

The antibiotic W341C, its ion transport properties and inhibitory effects on mitochondrial substrate oxidation.

We have examined the ion transport properties and the inhibition of rat liver mitochondrial substrate oxidation by the antibiotic W341C. W341C was able to transport 22Na+ and 42K+ across a bulk carbon tetrachloride layer. A preference was shown for K+ transport. With equal molar antibiotic concentrations, W341C transported 42K+ at a greater rate than the K+-selective ionophore nigericin, but transported 22Na+ at a lesser rate than the Na+-selective ionophore monensin. Like nigericin, W341C was able to deplete mitochondrial K+, but not Mg2+ nor Ca2+. The inhibition of mitochondrial substrate oxidation by W341C paralleled the patterns obtained with nigericin. These data indicate that W341C is a K+-selective ionophore that inhibits mitochondrial substrate oxidation by a mechanism analogous to that of nigericin.

Animals↗

PubMed1986

Chronic exercise training alters kinetic properties of rat skeletal muscle and myocardial lactate dehydrogenase.

10 weeks of treadmill running dramatically altered the kinetic properties of lactate dehydrogenase (EC 1.1.1.27) in skeletal muscle and myocardium in the rat. These changes, including Vmax, Km and optimal concentrations for pyruvate and lactate, indicate that exercise training induces the formation of H-isozyme in skeletal muscle and M-isozyme in the heart and represent metabolic adaptations of these tissues to chronic aerobic exercise.

Animals↗

PubMed1986

Stereochemical course of thiophosphoryl transfer catalyzed by cytosolic phosphoenolpyruvate carboxykinase.

Rat liver cytosolic phosphoenolpyruvate carboxykinase (PEPCK) utilizes inosine 5'-(3-thiotriphosphate) (ITP gamma S) as an excellent substrate, with Km and V values of 0.08 mM and 37 mumol min-1 (mg of protein)-1, respectively, compared with the corresponding values of 0.168 mM and 76 mumol min-1 (mg of protein)-1 for ITP. Thus, the V/Km values for the two substrates are the same. Reaction of (RP)-[gamma-18O2]ITP gamma S with oxalacetate catalyzed by cytosolic PEPCK produces (SP)-thio[18O]phosphoenolpyruvate. Therefore, thiophosphoryl transfer catalyzed by this enzyme proceeds with overall inversion of configuration at P. The reaction mechanism involves an uneven number of phosphotransfer steps, most likely a single step transfer between bound substrates. The results do not support the involvement of a phosphoryl enzyme intermediate in the mechanism.

Animals↗

PubMed1986

Methylisobutylxanthine blocks insulin antagonism of cAMP-stimulated glycogenolysis at a site distinct from phosphodiesterase. Evidence favoring an insulin-insensitive calcium release mechanism.

The widely used phosphodiesterase inhibitor MIX (1-methyl 3-isobutyl xanthine) blocked insulin antagonism of cAMP-stimulated glycogenolysis in rat hepatocytes but other phosphodiesterase inhibitors including Ro 20-1724 had no effect. Dose-response curves for MIX potentiation of cAMP-stimulated glycogenolysis and for MIX inhibition of the effects of insulin on cAMP-stimulated glycogenolysis suggested that at higher concentrations (250 microM) MIX may act at a site other than phosphodiesterase inhibition. MIX, at 250 microM, attenuated the insulin antagonism of glucose release stimulated by 8-bromo-cAMP, an extremely poor substrate for phosphodiesterase; other phosphodiesterase inhibitors did not. The possibility that MIX acts as an adenosine antagonist interfering with a postulated role for adenosine in insulin action was examined using N6-phenylisopropyladenosine (PIA), an Ra adenosine receptor agonist which increases hepatic cAMP levels. MIX inhibited insulin antagonism of PIA-stimulated glycogenolysis under conditions where it did not act as an adenosine antagonist (MIX and Ro 20-1724 both increased the response to PIA equally). The effect of concanavalin A on cAMP-stimulated glycogenolysis was antagonized by MIX, suggesting a post-receptor site of action for MIX. MIX paradoxically increased lactate production in the presence of 8-bromo-cAMP, reminiscent of the reported actions of calcium mobilizing hormones on lactate formation in fed hepatocytes. Cytosolic free Ca2+, as measured in Quin 2-loaded cells, was increased by MIX. In cells depleted of calcium, MIX no longer blocked insulin antagonism of 8-bromo-cAMP-stimulated glucose release, suggesting that MIX may function through an insulin-insensitive release of calcium. MIX greatly potentiated the stimulation of glycogenolysis by phenylephrine but did not alter the response to vasopressin. The relationship of this effect of MIX to the mechanism of insulin action and the ability of insulin to antagonize only alpha-adrenergic responses and not those of vasopressin is discussed.

1-Methyl-3-isobutylxanthine↗

PubMed1986

The effect of different types of physical exercise on glucose and citrulline synthesis in isolated rat liver parenchymal cells.

Rates of gluconeogenesis from lactate or pyruvate in hepatocytes from untrained rats were not increased by an acute (1 h) bout of exercise (running at 20 m/min). Hepatocytes from rats that had been exercise-trained for 1 month had lower rates of gluconeogenesis from lactate than cells from unexercised controls; the rates with pyruvate were identical. Hepatocytes from livers of trained animals immediately after 1 h of exercise synthesized glucose more rapidly and accumulated more citrulline than cells from resting rats.

Alanine↗

PubMed1986

The role of malate in exercise-induced enhancement of mitochondrial respiration.

Liver mitochondria isolated from rats immediately after exercise oxidize substrates more rapidly than do mitochondria from resting animals. In both fed and fasted rats, a 1-h period of exercise resulted in increased concentrations of malate in their livers and in the mitochondria isolated therefrom. This increase occurred in both untrained and exercise-trained rats. Because mitochondrial malate is known to facilitate mitochondrial uptake of other carboxylic substrates, it seems likely that the increased mitochondrial malate is responsible for the increased rate of oxidation. Rats injected with small amounts of malate (4.6 mumol/100 g body wt) yielded liver mitochondria with increased malate concentration and increased rates of oxidation of citrate, alpha-ketoglutarate, and succinate. The beta adrenergic antagonist propranolol (0.25 mg/100 g body wt) and the alpha 1 antagonist prazosin (same dose) did not abolish the effect of exercise on mitochondrial malate concentration or substrate oxidation.

Animals↗

PubMed1986

The role of malate in hormone-induced enhancement of mitochondrial respiration.

Shortly after the injection of glucagon, epinephrine, norepinephrine, vasopressin, or angiotensin II into fasted rats, mitochondria isolated from their livers contained elevated concentrations of malate and oxidized citrate, alpha-ketoglutarate, and, in some cases, succinate more rapidly than mitochondria from fasted, control rats. The administration of tryptophan, lactate, or ethanol and refeeding of rats fasted 24 h result in similar elevations of mitochondrial malate concentration and oxidation of added substrates. Treatments that resulted in elevated mitochondrial malate resulted also in increased uptake of added citrate, alpha-ketoglutarate, pyruvate, and, in some cases, succinate. It is postulated that the well-documented effect of gluconeogenic hormones on mitochondrial oxidation of carboxylic substrates may be mediated by malate which not only yields oxalacetate to support the tricarboxylic acid cycle but also facilitates the transport of added substrates, and which is regenerated in the tricarboxylic acid cycle.

Adenosine Diphosphate↗

PubMed1986

Enzymatic adaptation to physical training under beta-blockade in the rat. Evidence of a beta 2-adrenergic mechanism in skeletal muscle.

Nonselective and beta 1-selective adrenergic antagonists were tested for their effects on enzymatic adaptation to exercise training in rats as follows: trained + placebo (TC); trained + propranolol (TP); trained + atenolol (TA); and corresponding sedentary groups, SC and SP. Trained rats ran 1 h/d at 26.8 m/min, 15% grade, 5 d/wk, 10 wk. Both beta-antagonists were given at doses that decreased exercise heart rates by 25%. Training increased skeletal muscle citrate synthase, cytochrome c oxidase (Cyt-Ox), carnitine palmitoyltransferase (CPT), beta-hydroxyacyl coenzyme A dehydrogenase, mitochondrial malate dehydrogenase (MDH), and alanine aminotransferase (ALT) activities significantly in the TC group, but not in TP. These enzyme activities, except Cyt-Ox and CPT, were also significantly increased in TA. Hepatic phosphoenolpyruvate carboxykinase activity did not alter with training or beta-blockade. Fructose 1,6-bisphosphatase activity was lower in TC than in SC, but unchanged in TP or TA. Hepatic mitochondrial MDH and ALT activities increased with training only in TC. It is concluded that beta 2-adrenergic mechanisms play an essential role in the training-induced enzymatic adaptation in skeletal muscle.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1985

Multiple requirements for glycogen synthesis by hepatocytes isolated from fasted rats.

Glycogen synthesis from various combinations of substrates by hepatocytes isolated from rats fasted 24 h was studied. As reported by Katz et al. (Katz, J., Golden, S., and Wals, P. A. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3433-3437), appreciable rates of glycogen synthesis occurred only in the presence of gluconeogenic precursors and one of several amino acids, which includes L-glutamine. L-Leucine had negligible effects on glycogen synthesis from 20 mM dihydroxyacetone and/or 15 mM glucose when L-glutamine was not added to the medium. In the presence of 10 mM L-glutamine, L-leucine greatly increased glycogen synthesis from these substrates. alpha-Ketoisocaproate was ineffective, as was oleate. NH4Cl depressed glycogen synthesis from 10 mM glucose plus 20 mM dihydroxyacetone in the absence of added L-glutamine and enhanced that in its presence, but these effects were weak compared to those of L-leucine. The amino acid analogues L-norvaline and L-norleucine exerted effects that were similar to those exerted by L-leucine. Under all conditions studied, cycloheximide and puromycin inhibited net glycogen synthesis. Cycloheximide did not stimulate gluconeogenesis from dihydroxyacetone, or phosphorylase in hepatocytes from starved rats, or glycogenolysis in hepatocytes from fed rats. Puromycin, however, stimulated glycogenolysis in hepatocytes from fed rats. Glycogen synthesis from 20 mM dihydroxyacetone proceeds with a pronounced initial lag phase that can be shortened by incubation of cells with glutamine plus leucine before addition of dihydroxyacetone. Concurrent measurements of glycogen synthesis, glycogen synthase, and gluconeogenesis under different conditions reveal that in addition to protein synthesis, activation of glycogen synthase, which must occur to allow glycogen synthesis in hepatocytes, requires a second component which can be satisfied by addition of dihydroxyacetone or fructose to the cells.

Amino Acids, Branched-Chain↗

PubMed1985

Catecholamine and vasopressin stimulation of gluconeogenesis from dihydroxyacetone in the presence of atractyloside.

Atractyloside inhibited gluconeogenesis from dihydroxyacetone in hepatocytes from fasted rats and increased lactate synthesis. In the presence of atractyloside, lactate/pyruvate and beta-hydroxybutyrate/aceto-acetate ratios were increased and the accumulation of Fru-2,6-P2 was prevented. In the absence of atractyloside, gluconeogenesis from dihydroxyacetone was stimulated by dibutyryl-cAMP and, to a much lesser extent, by norepinephrine and vasopressin. Omission of Ca2+ increased the stimulation by norepinephrine but prevented that by vasopressin. High concentrations (greater than or equal to 40 microM) of atractyloside abolished the stimulation of gluconeogenesis by dibutyryl-cAMP but not that by norepinephrine or vasopressin. Exogenous Ca2+ was not required for hormonal stimulation in the presence of atractyloside. The stimulation by norepinephrine was inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N-tetraacetic acid or prazosin but not by propranolol. Atractyloside caused decreases of all glycolytic intermediates and an activation of pyruvate kinase. Norepinephrine partially reversed these effects. The mitochondrial and cytosolic ATP/ADP ratios were determined by digitonin fractionation of hepatocytes. Norepinephrine or vasopressin increased the cytosolic ATP/ADP in the presence of atractyloside. We suggest that the increased availability of cytosolic ATP could be responsible for the stimulation of gluconeogenesis by these hormones.

3-Hydroxybutyric Acid↗

PubMed1985

The antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action.

Insulin antagonized the lipolytic actions of epinephrine in rat epididymal adipocytes when the phosphodiesterase inhibitor, Ro 20-1724, was present. Adipocytes were depleted of functional cAMP by inhibiting adenylate cyclase with N6-phenylisopropyladenosine in the presence of adenosine deaminase such that Ro 20-1724 no longer stimulated lipolysis. The cAMP analogs 8-thioisopropyl-cAMP or 8-thiomethyl-cAMP, which are resistant to phosphodiesterase hydrolysis, were subsequently added to bypass adenylate cyclase and phosphodiesterase action. Under these conditions, insulin antagonized the lipolytic effects of these analogs, even in the presence of Ro 20-1724.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1985

Inhibition by 2,5-anhydromannitol of glycolysis in isolated rat hepatocytes and in Ehrlich ascites cells.

2,5-Anhydromannitol decreases lactate formation and 3H2O formation from [5-3H]glucose in isolated rat hepatocytes metabolizing high concentrations of glucose. The inhibition of glycolysis is accompanied by a slight decrease in the cellular content of fructose-6-P and a more substantial decrease in the cellular content of fructose-1,6-P2, with no change in the content of glucose-6-P. The 3H2O release data and changes in hexosephosphate distribution indicate possible inhibitions at phosphofructokinase-1 and phosphoglucose isomerase. 2,5-Anhydromannitol also inhibits glycolysis in Ehrlich ascites cells, but the tumor cells, unlike hepatocytes, must be treated with 2,5-anhydromannitol prior to exposure to glucose to obtain the inhibition. The decrease in 3H2O formation from [5-3H]glucose and the metabolite pattern that results from the addition of low concentrations (less than or equal to 0.25 mM) of 2,5-anhydromannitol indicate an inhibition at phosphofructokinase-1 that cannot be attributed to a decrease in the cellular content of fructose-2,6-P2. Higher concentrations (greater than or equal to 0.5 mM) of 2,5-anhydromannitol cause a substantial decrease in the cellular content of ATP that is accompanied by decreases in the content of glucose-6-P and fructose-6-P and transient increases in fructose-1,6-P2. In Ehrlich ascites cells, 2,5-anhydromannitol is metabolized to 2,5-anhydromannitol mono- and bisphosphate. The inhibition of glycolysis caused by 2,5-anhydromanitol decreases with time, because the phosphorylated metabolites formed during the preliminary incubation in the absence of glucose are rapidly dephosphorylated during the incubation in the presence of glucose.

Animals↗

PubMed1985

The structure of caltrin, the calcium-transport inhibitor of bovine seminal plasma.

The amino acid sequence of the bovine seminal protein, caltrin, which inhibits calcium transport into spermatozoa, has been determined. The protein contains 47 amino acid residues. Parts of the sequence are identical with that reported for bovine seminal plasmin, a protein possessing antibacterial activity. We believe the proteins are identical and that the previously reported sequence of seminal plasmin is in error.

Amino Acid Sequence↗

PubMed1985

A 31P NMR study of the epididymis and epididymal sperm of the bull and hamster.

31P NMR signals assigned to intracellular adenine nucleotides and to inorganic phosphate were detected in dense suspensions of epididymal sperm obtained from bulls or hamsters. Similar adenine nucleotide signals and an additional large resonance peak, attributable to extracellular glycerylphosphorylcholine, were observed with whole bovine cauda epididymides. Provision of the glycolytic substrate fructose to such sperm suspensions promoted apparent conversion of intracellular ADP to ATP with a concomitant decrease in cellular inorganic phosphate (Pi) content. Subsequent treatment with the methylxanthine caffeine resulted in diminution of the intracellular gamma-P-ATP signal that was consistent with the decreased ATP and ADP contents previously demonstrated by chemical analyses of cellular extracts. Alternatively, treatment with fructose followed by the membrane-selective detergent digitonin produced loss of the nucleotide NMR signals, indicating release of ATP and Pi from the sperm cytosol with subsequent hydrolysis in the extracellular medium. Comparison of intracellular Pi and ATP resonance signals with those of ATP and Pi in vitro, in media of varied pH and cation composition, allowed calculation of a cytosolic pH of 6.5-6.6 and a cytosolic Mg2+ concentration of 0.5 mM for fresh suspensions of bovine cauda epididymal sperm. Intracellular Pi of hamster epididymal sperm reported a similar cytosolic pH. Other, more acidic compartments were not detected in these experiments. However, during prolonged incubation, the pH of the bovine sperm interior slowly decreased as the extracellular medium was acidified by extensive production of lactate. Intracellular ATP was detectable until cytosolic pH declined to approximately 5.5. Rapid intracellular acidification, resulting from exchange of internal K+ for H+, was observed after treatment with carboxylic acid ionophore nigericin. This lowering of internal pH was followed by a slower return toward initial internal pH values, probably as a consequence of secondary exchange of internal protons for other external monovalent cations, rather than as a result of the operation of a cellular homeostatic mechanism. Together, these studies utilizing noninvasive NMR techniques provide evidence that within the bovine epididymis sperm utilize an unknown energy source to phosphorylate adenine nucleotides and maintain a slightly acidic cytosolic pH.

Animals↗

PubMed1985

Regulation of gluconeogenesis in hepatocytes from fasted alloxan-diabetic rats.

Hepatocytes from fasted, alloxan-diabetic rats were incubated in the absence of gluconeogenic substrates to deplete residual glycogen stores. Glucose production from lactate and pyruvate was enhanced in cells from diabetic rats relative to similarly treated hepatocytes from fasted, nondiabetic control rats. Gluconeogenesis from dihydroxyacetone, fructose, or glycerol was not increased but the formation of lactate plus pyruvate from dihydroxyacetone was decreased. The stimulation of gluconeogenesis by exogenous fatty acids was decreased by diabetes. The rates of gluconeogenesis in the presence of lactate plus pyruvate plus oleate were equal in hepatocytes from diabetic and control rats and indicate that the maximal rate of gluconeogenesis was not increased. With lactate plus pyruvate as substrates, stimulation of gluconeogenesis by norepinephrine or dibutyryl-cAMP was not altered by diabetes. The catecholamine stimulation of gluconeogenesis from glycerol also was unaffected. In contrast, diabetes decreased the maximal stimulation of gluconeogenesis from dihydroxyacetone by dibutyryl-cAMP, glucagon, or norepinephrine and this decrease was proportional to the decreased production of lactate plus pyruvate. The concentrations of glucagon or norepinephrine required for half-maximal stimulation were not altered by diabetes. Thus, the hormonal stimulation of gluconeogenesis from dihydroxyacetone is decreased by diabetes, probably because of decreased pyruvate kinase activity, but the interaction of glucagon and norepinephrine with hepatocytes and the subsequent stimulation of gluconeogenesis from physiologic substrates is not impaired.

Animals↗

PubMed1984

3-Aminopicolinate inhibits phosphoenolpyruvate carboxykinase in hepatocytes and increases release of gluconeogenic precursors from peripheral tissues.

3- Aminopicolinate , a hyperglycemic agent that activates purified phosphoenolpyruvate carboxykinase in the presence of Fe2+, inhibits glucose synthesis from lactate, pyruvate, asparagine, monomethyl succinate, or glutamine but does not affect that from fructose, dihydroxyacetone, sorbitol, or glycerol in hepatocytes isolated from rats fasted for 24 h. Lactate production from monomethyl succinate by hepatocytes is also inhibited by 3- aminopicolinate . This compound elevates the concentrations of pyruvate, malate, and aspartate but decreases that of phosphoenolpyruvate in hepatocytes incubated with lactate plus pyruvate. In rats, the ability of 3- aminopicolinate to elevate blood glucose concentration is unimpaired by renalectomy . The drug does not significantly affect glycemia in functionally hepatectomized rats but accelerates blood lactate and pyruvate accumulation to higher maximum concentrations even when kidney function is also ablated. It is concluded that 3- aminopicolinate inhibits phosphoenolpyruvate carboxykinase in hepatocytes, that the reported stimulation of renal glutaminase and glutamine gluconeogenesis by this compound does not contribute significantly to its hyperglycemic property, and that the drug increases gluconeogenic substrate supply from peripheral tissues.

Animals↗

PubMed1984

Site of insulin inhibition of cAMP-stimulated glycogenolysis.

Insulin antagonized the glycogenolytic actions of cAMP in isolated hepatocytes even in the presence of the phosphodiesterase inhibitors, 1-methyl-3-isobutylxanthine or d-4-(3-butoxy-4-methoxybenzyl)-2- imidizolidione . Stimulation of glucose release by 8-bromo-cAMP, which is not appreciably hydrolyzed by phosphodiesterase, was also inhibited by insulin in the presence of d-4-(3-butoxy-4-methoxybenzyl)-2- imidizolidione . This demonstrates that insulin can antagonize cAMP stimulation of glycogenolysis independent of possible effects of insulin on adenylate cyclase or phosphodiesterase. Under conditions where changes in cyclic adenine nucleotide concentrations (cAMP and 8-bromo-cAMP) were prevented, insulin depressed the 8-bromo-cAMP-stimulated protein kinase activity ratio. We conclude that phosphodiesterase activation is not required for insulin antagonism of cAMP-stimulated glycogenolysis. The effect of insulin under these conditions can be explained by an action of insulin on cAMP-dependent protein kinase independent of changes in cAMP levels.

1-Methyl-3-isobutylxanthine↗