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PubMed1979

Effect of thyroidectomy upon the activity of three mitochondrial shuttles in rats.

The activities of mitochondrial alpha-glycerophosphate, malate-asparte, and malate-citrate shuttles which oxidize NADH were determined in liver and kidney cortex mitochondria isolated from Tx and control rats. Weight gain, relative liver and kidney size, oxygen consumption, and serum thyroxine were also determined. Tx animals weighed less, had smaller livers and kidneys, and showed significantly lower oxygen consumption of the resting animal and a marked decrease in serum thyroxine levels. The activity of the three shuttles studies was significantly less in liver mitochondria from Tx animals, particularly when studied in the absence of added ADP. Thyroidectomy had less effect upon shuttle activity of mitochondria from the kidney cortex, with significant decrease in activity found only in the complete alpha-glycerophosphate shuttle and in the endogenous malate-aspartate and malate-citrate shuttles studied in the absence of ADP. Changes in shuttle activity caused by thyroid hormone are discussed in relation to other known metabolic effects of thyroxine, and the possible primary role of adenine nucleotide translocase activity in mediating other thyroxine-dependent metabolic changes is considered.

Adenosine Diphosphate↗
PubMed1982

Dynamic changes in the organophosphate profile of the experimental galactose-induced cataract.

Dynamic changes in lens organophosphate metabolites during 24 hr incubation in 30 mM galactose media were measured with phosphorus-31 nuclear magnetic resonance spectroscopy. The following phosphates were quantitated from the intact crystalline lens: adenosine triphosphate (ATP), adenosine diphosphate (ADP), inorganic orthophosphate, alpha-glycerophosphate, phosphorylated hexoses and trioses, nicotinamide adenine dinucleotide, uridine diphosphoglucose and uridine diphosphogalactose, glycerol-3-phosphorylethanolamine and 3-phosphorylcholine, and an unidentified phosphorus-containing molecule. The temporal sequences of metabolic events that define the dynamic rates of accumulation or depletion of lens organophosphates reveal that the first event in the decline of the tissue upon galactose incubation is a net consumption of ATP, which occurs as a sigmoidal function with time and which is typified by a characteristic half-life of 18 hr. Alpha-glycerophosphate accumulated at an increasing rate with time, whereas ADP, inorganic orthophosphate, and the other organophosphates were essentially unchanged. Cataract formation in the subcapsular and superficial cortical regions was visible after 16 hr incubation in the experimental buffer. These findings support the hypothesis that alterations in the organophosphate levels of the lens are contributing factors to the initial formation of the experimental galactose cataract.

Adenosine Diphosphate↗
PubMed1980

Mechanisms of increased brain glucose and glycogen after hydrocortisone: possible clinical significance.

We reported previously that chronic administration of hydrocortisone to normal developing mice increases the brain glucose content and cerebral energy reserve. The present report concerns possible mechanisms of this action. Increases in brain glucose (and glycogen) levels were not due to reduction of cerebral metabolic rate, and the effect of hydrocortisone in facilitating transport of hexose from blood to brain was not impressive. Chronic hydrocortisone treatment induced increases in the activities of brain glycerophosphate dehydrogenase and pyruvate carboxylase in vivo; there was no effect on eleven other enzymes of brain glucose and glycogen metabolism. In normal nursing mice, hydrocortisone produced consistent elevations in plasma beta-hydroxybutyrate (and glycerol) levels. Brain beta-hydroxybutyrate levels were also increased. Therefore, the brain glucose concentration may be elevated in these animals because of the availability of an increased supply of ketone bodies as alternative substrates for cerebral oxidative metabolism and biosynthesis. Ketonemia, elevated cerebral glucose and beta-hydroxybutyrate concentrations, and increased glycerophosphate dehydrogenase activity in brain suggest possible explanations for the beneficial action of adrenocorticotropic hormone and glucocorticoids in the treatment of infantile myoclonic epilepsy and other neurological disorders.

Animals↗
PubMed1993

Enzymatic, metabolic and secretory perturbations in pancreatic islets of thyroidectomized rats.

In thyroidectomized rats, the activity of FAD-linked glycerophosphate dehydrogenase was severely diminished in liver homogenates but not affected significantly in pancreatic islet homogenates, whilst the activity of 2-ketoglutarate dehydrogenase was decreased modestly in both liver and islet homogenates. Likewise, in intact islets of thyroidectomized rats, the generation of 3HOH from [2-3H]glycerol was not decreased, and the ratio between oxidative and total glycolysis not significantly lower than in islets from sham-operated rats, at least in the presence of a high concentration of D-glucose. Nevertheless impaired oxidation of both D-[3,4-14C]glucose and D-[6-14C]glucose was observed in islets of thyroidectomized rats, the relative magnitude of such a decrease being more pronounced at a low than at a high D-glucose concentration. Such metabolic anomalies coincided with a lower level of plasma insulin and decreased output of insulin by islets incubated at low (2.8 mM), but not higher, concentrations of D-glucose. It is concluded that hypothyroidism does not mimic the deficiency in islet FAD-linked glycerophosphate dehydrogenase activity found in rats with inherited or acquired non-insulin-dependent diabetes.

Blood Glucose↗
PubMed1994

Cellular and matrix changes before and at the time of calcification in the growth plate studied in vitro: arrest of type X collagen synthesis and net loss of collagen when calcification is initiated.

To understand the growth, maturation, and regulation of growth plate chondrocytes, it is necessary to isolate the different chondrocytes into distinct subpopulations of maturational development. Five subpopulations (A-E) of bovine fetal growth plate chondrocytes were separated by discontinuous gradient centrifugation. Four subpopulations (B, C, D, and E, from low to high density) with good viability were cultured at high density in microwells for up to 30 days. They all established an extensive extracellular matrix composed of proteoglycan and collagen. The largest and last dense cells in subpopulation B were the first to synthesize (at days 5-6) type X collagen and to calcify this matrix. Matrix calcification (formation of hydroxyapatite in the presence of sodium beta-glycerophosphate) always followed the initiation of type X synthesis. All the other subpopulations synthesized type X collagen and calcified their extracellular matrix. Although these events occurred in the same order, they were delayed according to the order of increasing cell size. These observations indicate that these subpopulations represent different stages in cellular maturation that lead to expression of the hypertrophic phenotype. Once mineral formation was well established, there was an increase in the matrix content of the C-propeptide of type II collagen (which is known to bind to hydroxyapatite and accumulate in calcifying extracellular matrix). This was accompanied by a reduction in the total collagen content, which accompanied an abrupt reduction in type X collagen synthesis, whereas type II collagen synthesis was largely maintained. These reductions in collagen content and type II collagen synthesis were not observed in the absence of calcification (beta-glycerophosphate omitted from culture). This new culture system recreates many of the sequential cellular and extracellular changes exhibited in situ during the development of the physis and provides new information about cellular and extracellular matrix changes that occur before and at the time of calcification.

Absorptiometry, Photon↗
PubMed1980

A study of binding-solubilization of some glycolytic enzymes in striated muscle in situ.

The solubilization of lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and alpha-glycerophosphate dehydrogenase (GAPDH) was studied in pressed muscle as a function of ionic strength and NADH concentration. The results indicate that these factors affect the binding-solubilization of LDH and GAPDH in a similar way to their effect in dilute homogenized tissue. Alpha-glycerophosphate dehydrogenase was included as a typical soluble enzyme, since we have been unable to demonstrate its binding to subcellular fractions under any conditions. As with homogenized tissue, LDH was less susceptible to solubilization by ionic strength than GAPDH. It was demonstrated that LDH isozymes richer in heart-type subunits were more easily removed from muscle by centrifugation-imbibition than isozymes richer in the muscle-type subunits. This was interpreted as indicating that binding of the enzyme to subcellular structures was a major factor in the restricted removal of these enzymes from muscle, since only the muscle-type subunit is capable of binding to subcellular particles. It was further demonstrated that LDH could be taken up into muscle tissue, depleted in the enzyme, against an apparent concentration gradient. This was also interpreted as binding of the enzyme to the particulate structure of the muscle. Furthermore, this uptake of LDH occurred under conditions of ionic strength (0.25) and pH (7.5) that would prevent binding of the enzyme to the particulate fraction of a dilute suspension of homogenized muscle tissue. Thus, physiological conditions of pH and ionic strength do not necessarily induce solubilization of chicken breast muscle LDH in situ. Data obtained with dilute tissue homogenates, therefore, may not necessarily be easily and safely extrapolated to conditions in situ.

Animals↗
PubMed1986

Sensitivity of preadipose 3T3 cells to growth hormone.

Cultured preadipose 3T3 cells are able to undergo a process of differentiation through which they are converted into adipose cells. Growth hormone induces this conversion in resting cultures but not in growing cultures. It was of interest to determine the period of cell sensitivity to the hormone and the timing of the induction of glycerophosphate dehydrogenase, a key enzyme in lipogenesis. It was found that 3T3-F442A cells became highly sensitive to rat growth hormone at confluence but that high sensitivity remained for only 3 days; thereafter, the responsiveness to the rat growth hormone declined rapidly. Refeeding of the cells with fresh medium did not lead to the recovery of the hormone sensitivity, indicating that the decrease in sensitivity was not due to exhaustion of medium components but that it seemed to be a specific property of F442A cells. As glycerophosphate dehydrogenase activity was detected at nearly the same time as its mRNA was measurable, it is likely that the mRNA is translated immediately after its synthesis.

Adipose Tissue↗
PubMed2004

Stereospecific effects of catechin isomers on insulin induced lipogenesis in 3T3-L1 cells.

We studied the influence of (+)- and (-)-catechin contained in green tea on insulin induced lipogenesis in 3T3-L1 cells. In 14 days of culture with insulin, the intracellular triglyceride concentration and the activities of glycerophosphate dehydrogenase, a marker of adipose conversion, increased. The addition of 0.02 mg/ml (+)-isomer stimulated the accumulation of triglyceride induced by insulin, but the addition of the same concentration of (-)-isomer inhibited lipogenesis. The activities of glycerophosphate dehydrogenase were changed by (+)- and (-)-catechin in the same direction as the corresponding changes in triglyceride accumulation. These data suggest a biological significance of catechins, with opposite effects on lipid metabolism depending on the isomer.

3T3 Cells↗
PubMed1993

Molecular analysis of lipid macroamphiphiles by hydrophobic interaction chromatography, exemplified with lipoteichoic acids.

Analyzed were (I) the oligoglucosyl-, alanyl-substituted poly(glycerophosphate) lipoteichoic acid of Enterococcus hirae containing Glc(alpha 1-2)Glc(alpha 1-3)acyl2-Gro and a phosphatidyl derivative thereof as lipid anchor, (II) the poly(digalactosyl, galactosylglycerophosphate) lipoteichoic acid of Lactococcus garvieae containing the same glycolipid and an acyl derivative of it, and (III) the N-acetylglucosaminyl-, alanyl-substituted poly(glycerophosphate)lipoteichoic acid of Staphylococcus aureus with Glc(beta 1-6)Glc(beta 1-3)acyl2Gro as the sole lipid moiety. Hydrophobic interaction chromatography on octyl-Sepharose separated lipoteichoic acids I and II into two peaks according to the number of fatty acids. Within each peak further fractionation occurred in the order of decreasing length of the hydrophilic chain. A similar fractionation was observed within the single peak of lipoteichoic acid III. With lipoteichoic acid I and III the extent of glycosylation decreased with decreasing length of the hydrophilic chain whereas the content of alanine ester remained either constant or increased. Variations in the oligosaccharide pattern of lipoteichoic acid I and in the fatty acid composition of all lipoteichoic acids could also be observed. Collectively, the data provide for the first time a detailed picture of the complex polydispersity of lipoteichoic acids comprising the number of fatty acids, the length of the hydrophilic chain, the kind and extent of chain substitution, and the fatty acid composition. The procedure will also be applicable to the molecular analysis of lipopolysaccharides and bacterial lipoglycans. Moreover, the results are of physicochemical interest because they demonstrate for lipid macroamphiphiles an inverse relationship between hydrophobicity and the size of the hydrophilic headgroup.

Animals↗
PubMed1978

Studies on some thermogenetic enzymes in brown adipose tissue of genetically obese mice.

The effects of cold exposure on several enzymes in brown adipose tissue (BAT) were examined in two types of genetically obese mice (C57BL/6J-ob and C57BL/6J-Ay) and their thin litter mates. In the thin C57BL/6J mice, the activities of two alpha-glycerophosphate-generating enzymes i.e., glycerokinase (GK) and alpha-glycerophosphate dehydrogenase (GDH), increased in BAT at cold exposure. Resemble results were observed in the yellow obese mice (A(y) mice). Nevertheless, in the obese-hyperglycemic mice (ob mice) showing a poor resistancy to cold, the increases of these enzymes were not observed. In addition, the increase of lipase activity, observed in BAT of the thin litter mates, was also not observed in BAT of ob mice. The decrease of NEFA release from BAT at cold exposure and the prominentry high inhibition rate of lipase by M NaCl were observed also in ob mice. Then the qualitive change of BAT lipase may exist in ob mice.

Adipose Tissue, Brown↗
PubMed1990

Oral magnesium reduces ventricular ectopy in digitalised patients with chronic atrial fibrillation.

We have examined the effects of magnesium replacement therapy upon post-exercise heart rate and incidence of ventricular premature beats (VPB) in digitalised patients with AF. In 11 such patients, all of whom had serum magnesium concentrations of less than 0.85 mmol/l, treatment with magnesium glycerophosphate was associated with a significant reduction in number of VPBs (982 v. 416 VPB/24 h). Five patients had a high prevalence of ventricular ectopy (greater than 300 VPB/24 h) and these subjects showed particularly marked decreases in VPBs during magnesium treatment (1998 v. 690 VPB/24 h). Three patients had slightly increased QTc intervals but these did not change during magnesium replacement. No significant changes were seen in the mean post-exercise heart rate although 2 subjects did show falls of 25% or more during magnesium replacement. We conclude that treatment with magnesium glycerophosphate may be associated with a decreased prevalence of ventricular ectopy in some digitalised patients with chronic AF and mild-moderate hypomagnesaemia.

Aged↗
PubMed1977

Cytochrome c oxidase activity in T/t6 (balanced lethal) mutant mice.

R-1 (1450g) and R-2 (25,000g) liver fractions from T/t6 and B6CBAF1 hybrid mice were analyzed for their protein content, mitochondria concentrations, and activities of three respiratory-chain enzymes of the mitochondrial inner membrane: cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, E.C. 1.9.3.1.), alpha-glycerophosphate dehydrogenase [L-glycerol-3-phosphate: (acceptor) oxidoreductase, E.C. 1.1.99.5], and succinate-cytochrome c reductase. Only cytochrome c oxidase activity, calculated as units per 10(10) mitochondria, was significantly lower in both R-1 and R-2 fractions of T/t6 mice. Cytochrome c oxidase activity varied greatly among T/t6 mice, as did their liver mitochondria concentrations and body weights. Cytochrome c oxidase activity in the R-1 fraction of T/t6 mice, averaged about 40% lower than in B6CBAF1 mice. alpha-Glycerophosphate dehydrogenase activity was often elevated in T/t6 mice, particularly in the R-2 fraction. The T/t locus, a complex genetic locus on chromosome 17, may contain genes important to the function and biogenesis of mitochondria.

Age Factors↗
PubMed1976

Adenosine triphosphate hydrolysis in rat dental tissues. A histochemical study of ion dependencies.

The effect of EDTA-decalcification, reactivating and activating procedures on the hydrolysis of ATP was studied histochemically in developing dental tissues in the rat. The incubation media contained lead citrate at alkaline pH and lead nitrate at neutral pH, and the results with ATP as substrate were compared with those obtained with beta-glycerophosphate. The ion dependency of ATP hydrolysis could only be ascertained in decalcified sections. As in earlier studies on the hydrolysis of beta-glycerophosphate in dental tissues, this hydrolysis could readily be reactivated through preincubation of the sections in a series of 0.1 M solutions of divalent cations; Zn2+ being the most efficient. This treatment was now found also to give rise to an ATP hydrolysis, which occurred without the need for activating ions in the incubation medium. This ATP hydrolysis should thus be described as nonspecific and, in terms of ion dependency, as due to a metalloenzyme, i.e. alkaline phosphatase. Activating ion dependent ATP hydrolysis in the dental tissues was found in the blood vessels and in the apical part of the secretory ameloblasts. The former was activated by Mg2+, Ca2+ and Mn2+, and the latter by Ca2+ and--almost specifically--by Sr2+. Preincubation with Zn2+ always inhibited the ion dependant ATP hydrolysis in the dental tissues.

Adenosine Triphosphatases↗
PubMed1981

Ultracytochemical localization of adenylate cyclase activity in rat thymocytes.

Ultrastructural localization of adenylate cyclase (AC) activity was investigated in suspensions of unfixed isolated rat thymocytes using a medium containing 0.6 mM 5'-adenylylimidodiphosphate (AMP-PNP) as a substrate, 10 mM MgSO4 as an activator, 5 mM theophylline as an inhibitor of 3' 5'-AMP-phosphodiesterase and 2 mm lead nitrate as a capturing agent. AC activity was demonstrated in plasma membrane, perinuclear space, endoplasmic reticulum, Golgi complex, centriole microtubules and mitochondria. AC was activated with 10(-4) M adrenaline in the presence of 5'-guanylylimido-diphosphate (GMP-PNP) as well as with 10(-2) M NaF. In the cells incubated in a medium devoid of theophylline and containing 5'-AMP instead of AMP-PNP, 5'-nucleotidase activity was observed in the same cell structures as AC activity, Hydrolysis of 5'-AMP in the nucleus was much stronger than that of AMP-PNP. 10 mM NaF markedly inhibited hydrolysis of 5'-AMP in all cell structures. No staining was observed with 2 mM beta -glycerophosphate as a substrate. Incubation of unfixed thymocytes in media containing AMP-PNP, 5'-AMP or p-nitrophenyl phosphate, but not beta -glycerophosphate, induced both in the nucleus and in the cytoplasm in some cells an appearance of a transitory reticular formation consisting of about 303nm thick strands which could penetrate the nuclear envelope and plasma membrane and form connections with adjacent cells. The transitory reticular formation seems to belong to the cytoskeleton and to be involved in cell aggregation.

Adenylyl Cyclases↗
PubMed1980

The effect of thyroid hormone on mitochondrial biogenesis and cellular hyperplasia.

The purpose of this investigation was to study the effects of thyroid hormone treatment on the levels of DNA, RNA, and protein in hepatocytes and hepatocyte mitochondria. A preliminary investigation was conducted to establish an effective dosage of thyroid hormone. Male Sprague-Dawley rats were given daily subcutaneous injections of L-thyroxine (20, 40, or 60 micrograms/100 g body weight) and the following determinations made over a 14-day period: (1) body weight; (2) total body respiration; and (3) the activities of the mitochondrial enzymes, succinate dehydrogenase and alpha-glycerophosphate dehydrogenase. Dosages of 20 and 40 micrograms L-thyroxine/200 g body weight produced significant stimulation of (a) total body respiration and (b) succinate dehydrogenase and alpha-glycerophosphate dehydrogenase activities without any inhibitory effects on normal weight gain of the animals. Injections of 40 micrograms L-thyroxine/100 g body weight were utilized for subsequent studies. Hepatic DNA levels of treated animals were greater than age-paired control values by 28% on day 7 and 43% by day 14. Total liver RNA levels of thyroid-treated animals were 17% greater than those of controls by day 7 and 47% greater by day 14. Analyses were also performed on mitochondria quantitatively collected by rate zonal centrifugation. Total liver mitochondrial DNA levels in thyroid-treated animals were greater than age-paired controls by 79% at 7 days but only 67% at 14 days since a small gain occurred in control animals and no further increase occurred in treated rats during the second week. Mitochondrial RNA and protein from treated livers were 26% and 16% higher, respectively, than age-paired controls at day 7 and 40% and 58% higher, respectively, at day 14. The results of this study indicated that thyroid hormone treatment produces hyperplasia and an increase in mitochondrial number and mass in rat liver.

Animals↗
PubMed1988

Quantitative enzyme histochemistry of rat foetal brain and trigeminal ganglion.

The increasing concern and the efforts in determining neurological effects in offsprings resulting from maternal exposure to xenobiotics are faced with several difficulties in monitoring damage to the central nervous system. In this paper, the efficiency of several enzyme histochemical reactions for analysing the forebrain and the trigeminal ganglia of rat foetuses are reported. Brains of 20-day-old Sprague-Dawley rat foetuses were frozen and analysed for 18 enzymes that had previously been used to monitor initial injury caused by toxic compounds in liver and other organs. Eight enzymes appeared suitable as histochemical markers for the functional integrity of different areas in brain and ganglia of rats exposed to xenobiotics. They were lactate, malate, glycerophosphate (NAD-linked), succinate, aldehyde and glucose 6-phosphate dehydrogenases, alpha-glycerophosphate-menadione oxidoreductase and cytochrome c oxidase. The activities of the enzymes were determined by microphotometry and the arrangement of absorbances of the enzyme final reaction products into appropriate analytical tables is proposed as an efficient procedure for data analysis.

Acetylcholinesterase↗
PubMed1979

Some observations on the phosphatase cytochemistry of the submandibular gland of cat.

The localization of reaction product arising from incubation with thiamine pyrophosphate at pH 7.2 and beta-glycerophosphate or nitrophenylphosphate at pH 5 has been examined in central and demilunar acinar cells, myoepithelial cells, and cells of the striated ducts. Thiamine pyrophosphate reaction product was seen in the inner saccules of the Golgi apparatuses of the different types of cell. beta-Glycerophosphate and nitrophenylphosphate reaction products were seen in lysosomes, and in GERL-like structures in all except the cells of the striated ducts, in which the reaction products were observed in the Golgi apparatus. In central cells, the latter two reactions products were also seen in secretory granules and the cytosol.

Animals↗