Renewal of glycerol in the visual cells and pigment epithelium of the frog retina.

The renewal of glycerol in the visual cells and pigment epithelium of the frog retina was studied by autoradiographic analysis of animals injected with [2-(3)H]glycerol. Assay of chloroform:methanol extracts showed that the labeled precursor was used mainly in lipid synthesis, although there was also some utilization in the formation of protein. Radioactive glycerol was initially concentrated in the myoid portion of rods and cones, indicating that this is the site of phospholipid synthesis in visual cells. The glycogen bodies (paraboloids) of accessory cones were also heavily labeled, suggesting the diversion of some glycerol into glycogenic pathways. In the pigment epithelium, only the oil droplets became significantly radioactive. The outer plexiform layer (which contains the visual cell synaptic bodies) and the cone oil droplets gradually accumulated considerable amounts of labeled material. Within 1-4 h, labeled molecules began to appear in the visual cell outer segments, evidently having been transported there from the myoid portion of the inner segment. Most of these were phospholipid molecules which became distributed throughout the outer segments, presumably replacing comparable constituents in existing membranes. In rods only, there was also an aggregation of labeled material at the base of the outer segment due to membrane biogenesis. These highly radioactive membranes, containing labeled molecules of lipid and protein, were subsequently displaced along the rod outer segments due to repeated membrane assembly at the base. The distribution of radioactivity supported the conclusion that membrane renewal by molecular replacement is more rapid for lipid than it is for protein.

Lipolysis and lipogenesis from glucose in human fat cells of different sizes. Effects of insulin, epinephrine, and theophylline.

Lipogenesis from glucose and lipolysis in human omental and subcutaneous fat cells were studied as functions of adipose cell size and number in adult females. Since subcutaneous fat cells were larger than those prepared from the greater omentum, a comparison could be made of the metabolism of different sizes of cells within individual subjects. Rates per cell of glyceride-glycerol and glyceride-fatty acid synthesis from glucose were similar in omental and subcutaneous fat cells incubated in the presence or absence of insulin. However, subcutaneous fat cells exhibited higher rates of basal lipolysis than omental fat cells and these differences were maintained when lipolysis was stimulated with theophylline. Different rates of lipolysis were not demonstrable after incubations with epinephrine, indicating that subcutaneous fat cells were less responsive to this hormone than smaller omental fat cells. Correlation and partial correlation analysis showed that differences in basal and theophylline-stimulated lipolysis between fat cells prepared from different subjects and between omental and subcutaneous fat cells could be accounted for by differences in adipose cell volume. In subcutaneous fat cells highly significant intercorrelations were demonstrated between cell volume, basal lipolysis, and the basal conversion of glucose to glyceride-glycerol. There was no correlation between fat cell volume, age, or relative obesity and the effects of theophylline or insulin on lipolysis or lipogenesis from glucose in vitro when the data were expressed as percentage changes above basal values.

Biosynthesis of acyl groups on molecular species of biliary phosphatidylcholines during metabolism of [2,2,2-2H3]ethanol.

Incorporation of deuterium into different positions of individual molecular species of biliary phosphatidylcholines was determined in bile fistula rats given [2,2,2-2H3]ethanol under conditions ensuring maximal rate of oxidation for 24 h. The deuterium-labelling of the glycerol moiety of the major molecular species was about 6-8 atom% at the end of ethanol administration. The deuterium excess at each of the different positions of the glycerol moiety of 1-palmitoyl-2-linoleoyl phosphatidylcholine was less than 3 atom%. From the isotopic composition of the palmitoyl residues of the phosphatidylcholines, it was calculated that [2,2,2-2H3]ethanol supplied about 35-40% of the acetyl-CoA forming the terminal methyl group and about 25-30% of the other C2 units of the palmitic acid chain. This difference in deuterium incorporation was interpreted as being due to an isotope effect, probably in the rate-limiting carboxylation step of acetyl-CoA. Most or perhaps all of the acetyl groups derived from ethanol were introduced into the terminal methyl group without loss of deuterium. This indicates that citrate is not an important carrier of acetyl-CoA in the biosynthesis of fatty acids from ethanol.

Differential labeling of glycerol moieties of phospholipids and triacylglycerols of cultured mammalian cells by [U-14 C] glucose.

Rabbit liver cells, in which fatty acid synthesis was suppressed by the rabbit serum component of the medium, were grown through 8- to 120-fold increases in cell numbers and mass of cell lipid in the presence of [U-14 C]-glucose. Triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine were isolated from the total cell lipid and deacylated. Carbons 1 and 3 of the glycerol from the triacylglycerols and the no. 1 glycerol carbons of the two deacylated phospholids were oxidized by periodate and isolated as the dimedon derivative of formaldehyde. The specific activities of the glycerol carbons indicated that 58, 44, and 37 percent of the glycerol of the triacylglycerols. phosphatidylcholine, and phosphatidylethanolamine, respectively, were derived from the glucose of the medium. An additional 8 percent and 1-2 percent of the glycerol of each lipid was derived, respectively, from [U-14 C] glycerol and U14C-labeled amino acids added to the medium. In agreement with an experiment with albumin-bound [9,10- minus 3H]-oleic acid, and with smilar earlier experiments, it appears likely that appriacylglycerols originated from serum lipoproteins, or their partial hydrolysis products. An appreciable part of the ethanolamine of the cells' phosphatidylethanolamine originated from exogenous U- minus 14 C-labeled amino acids. Phosphatidyl-ethanolamine, however, was not a primary source of phosphatidylcholine. Labeling of the fatty acids of triacylglycerols and phospholipids by radioactive glucose, glycerol and amino acids was negligible.

Formation of ether lipids and wax esters in mammalian cells. Specificity of enzymes with regard to carbon chains of substrates.

The incorporation of radioactivity from individual constituents of an equimolar mixture of saturated straight-chain alcohols (14:0, 16:0, 18:0, 20:0) and of nearly uniform mixtures of isomeric cis- or trans-octadecenols (delta 8-delta 16) into alkyl, alk-1-enyl and acyl moieties of diradylglycerophosphocholines and diradylglycerophosphoethanolamines and into alkyl and acyl moieties of wax esters was studied in rat brain as well as in L 1210 and S 180 ascites cells. The pattern of incorporation of radioactivity from the substrates into alkyl and alk-1-enyl moieties of ether phospholipids and into alkyl moieties of wax esters reveals the following: (1) The enzymes catalyzing the biosynthesis of alkylacylglycerols, the common intermediates of cholinephospholipids and ethanolaminephospholipids, have no substrate specificity with regard to position of the double bond of either cis- or trans-octadecenols or of intermediate ether lipids derived therefrom. (2) CDPcholine:diradylglycerol cholinephosphotransferases exhibit a strong preference for alkylacylglycerols with cis-8, cis-9 and cis-10-octadecenyl moieties, but no preference for the double bond position in the trans-octadecenylacylglycerols. (3) CDPethanolamine:diredylglycerol ethanolaminephosphotransferases have no substrate specificity with regard to position of the double bond in cis- or trans-octadecenyl moieties of alkylacylglycerols. (4) THe enzyme systems catalyzing the biosynthesis of alkylacylglycerophosphocholines and alkylacylglycerophosphoethanolamines exhibit substrate specificity with regard to chain-length of saturated alcohols and intermediate ether lipids derived therefrom. (5) Alkylacylglycerophosphoethanolamine desaturase and (6) wax ester synthase are highly specific for alkylacylglycerophosphoethanolamines and long-chain alcohols, respectively, with regard to chain-length of saturated alkyl moieties, but not with regard to position of double bonds of cis- or trans-octadecenyl moieties.

Inhibition of phosphoenolpyruvate carboxykinase, glyceroneogenesis and fatty acid synthesis in rat adipose tissue by quinolinate and 3-mercaptopicolinate.

To study the interrelationships of phosphoenolpyruvate carboxykinase and glyceroneogenesis in adipose tissue, investigations with two effectors of the hepatic carboxykinase, Fe2+ and Mn2+, and two inhibitors of the enzyme and of gluconeogenesis in liver, quinolinic acid and 3-mercaptopicolinic acid, were carried out. Incubating adipose tissue cytosol with 30 microM Fe2+ or 100 microM Mn2+ prior to assaying for phosphoenolpyruvate carboxykinase activity doubled the enzyme activity. Inhibition of the enzyme by quinolinate alone was minimal. Adding 30 microM Fe2+ to the cytosol decreased the K0.5 (concentration that gives 50% inhibition) for quinolinate to 0.4 mM and the K0.5 for mercaptopicolinate from 200 to 14 microM. Activating the enzyme with 100 microM Mn2+ did not lower the K0.5 values and adding 500 microM Mn2+ to the cytosol completely interfered with the enhancement of inhibition induced by Fe2+. Each inhibitor interfered with 14C incorporation into glyceride glycerol from labeled pyruvate, alanine and lactate in suspensions of adipocytes. Adding 1 mM Mn2+ to the adipocyte suspension almost completely prevented the inhibition of pyruvate and alanine incorporation into glyceride glycerol, but adding the Mn2+ or 250 microM Fe2+ to the adipocytes in the absence of inhibitors did not enhance glyceride glycerol formation. Adding 250 microM Fe2+ to the adipocytes did not enhance inhibition of lipid synthesis by mercaptopicolinate or quinolinate. Mercaptopicolinate did not inhibit glyceride glycerol, fatty acid, total lipid or CO2 production from glucose. The lack of activation of glyceride glycerol synthesis by added Fe2+ or Mn2+, the lack of enhancement of pyridine carboxylate inhibition by Fe2+ and the interference with inhibition by Mn2+ are compatible with the idea that a transition metal ion similar to Fe2+, if not Fe2+ itself, is available to, or loosely bound to, the adipose tissue carboxykinase in vivo. Taken together with the results of previous work which showed ferroactivator (a cytosol protein necessary for Fe2+ activation of the carboxykinase) to be present in adipose tissue, the present results indicate that the control of the adipose tissue carboxykinase may be similar to the enzyme in liver. Fatty acid synthesis was also diminished by the inhibitors, albeit to a lesser extent than was glyceride glycerol formation. It is hypothesized that this was secondary to decreased esterification caused by the lack of glycerol 3-phosphate from inhibition of the carboxykinase. Decreased esterification would lead to a build-up of fatty acyl CoA which inhibits fatty acid synthesis.

Effect of methoxyindole 2-carboxylic acid and 4-pentenoic acid on adipose tissue metabolism.

1. Administration of methoxyindole 2-carboxylic acid to rats caused an increase in circulating free fatty acids which was associated with rapid hypoglycemia in fasted rats and liver glycogenolysis without hypoglycemia in fed rats. 2. The incorporation of labeled glucose, pyruvate and acetate carbons into triacylglycerol-glycerol, triacylglycerol-fatty acids and CO2 was inhibited in epididymal fat pads from methoxyindole 2-carboxylic acid-treated rats and by the addition of methoxyindole 2-carboxylic acid in vitro. In contrast, palmitate esterification and oxidation were enhanced by methoxyindole 2-carboxylic acid. 3. The activity of enzymes associated with fatty acid synthesis was reduced to a varying degree in the presence of methoxyindole 2-carboxylic acid in the reaction mixture in concentrations lower than those used to inhibit glucose and pyruvate metabolism in the intact tissue in vitro. 4. 4-Pentenoic acid, a potent inhibitor of pyruvate and palmitate metabolism in the liver, was considerably less effective in adipose tissue. 5. The effect of the two hypoglycemic substances investigated on adipose tissue metabolism seems to be different.

The carbon pathway for lipogenesis in isolated adipocytes from rat, guinea pig, and human adipose tissue.

The synthesis of fatty acids from a variety of labeled substrates by isolated adipocytes of the rat, guinea pig, and human was investigated. The incorporation of radioactive glucose and pyruvate into triglyceride fatty acids was considerably lower in human than either rat or guinea pig adipose tissue. By contrast, the incorporation of palmitate into adipose tissue triglycerides was approximately the same in all three species. End carbon analysis of fatty acids isolated from adipocytes incubated with pyruvate-U-14C indicated that although the synthesis of fatty acids in human adipose tissue was extremely low compared to that of the rat and guinea pig, it represented de novo biosynthesis rather than chain elongation of existing fatty acids. It is suggested that in the human, fatty acids are synthetised de novo primarily in the liver. In adipose tissue, lipogenesis consists essentially of the esterification of fatty acids, obtained from plasma, into triglycerides.

Glycerol, a metabolic end product of Trichomonas vaginalis and Tritrichomonas foetus.

Glycerol was demonstrated as an end product of anaerobic glucose metabolism in Trichomonas vaginalis and Tritrichomonas foetus, produced in addition to acetate, H2, CO2, and lactate or succinate. In T. vaginalis strain C-1, glycerol amounted to 16% of the fermentation products and was formed at an average rate of 38 nmol min-1 (mg protein)-1. Corresponding figures for T. foetus strain KV1 were 7% and 4.8 nmol min-1 (mg protein)-1. The amounts of glycerol detected compensated almost exactly for the deficits in fermentation products recognized earlier, thus complete redox balances can now be provided for both organisms. The metronidazole-resistant T. foetus strain KV1-1MR-100 excreted only negligible amounts of glycerol and carried out an ethanol-CO2 fermentation. Aerobiosis hardly affected glycerol formation in T. vaginalis strains C-1 and NYH 286, but almost completely abolished it in T. foetus strain KV1. An NADP-dependent glycerol 3-phosphate dehydrogenase and a Mg2+-dependent glycerol 3-phosphatase were detected in the cytosol of both species. The phosphatase is distinct from the particle-bound nonspecific acid phosphatase. Glycerol kinase activity was not detected in either organism. Enhanced pCO2 did not affect the ratio of fermentation products in T. vaginalis strain C-1, but significantly increased the amount of succinate, and decreased the amounts of acetate, H2, and CO2, formed by T. foetus.

A comparative study of D-lactate, L-lactate and glycerol formation by four species of Leishmania and by Trypanosoma lewisi and Trypanosoma brucei gambiense.

Leishmania braziliensis panamensis, L. donovani, L. major, and L. mexicana amazonensis promastigotes, Trypanosoma lewisi bloodstream forms, and T. brucei gambiense procyclic forms were incubated with glucose as sole carbon source. All species consumed glucose more rapidly under aerobic than anaerobic conditions. All produced glycerol under anaerobic conditions, though the rate of glycerol production by T. lewisi was markedly lower than that by the other species. The four Leishmania species produced D-lactate, but not L-lactate, whereas T. b. gambiense procyclic forms produced L-lactate, but not D-lactate, and T. lewisi produced both isomers.

Insulin binding and action on adipocytes from female rats with experimentally induced chronic hyperprolactinemia.

We studied insulin binding and action in adipocytes from female rats with chronic hyperprolactinemia induced by grafting an anterior pituitary gland under the right kidney capsule. Normal basal insulin plasma levels were detected. An increase in insulin binding due to an increased number of receptors was observed (grafted: 193,000 +/- 13,000 (6) receptors/cell vs. controls: 136,000 +/- 17,000 (6) receptors/cell, P less than 0.05). No changes in receptor affinity were detected (ED50 grafted: 2.3 X 10(-9) M and ED50 controls: 1.6 X 10(-9) M). The antilipolytic activity of insulin was significantly decreased in adipocytes from rats with hyperprolactinemia, indicating an insulin-resistant state in these animals. These findings suggest that the chronic hyperprolactinemic state can modify receptor and post-receptor insulin events in rat parametrial adipose tissue.

NAD-glycohydrolase activity of botulinum C2 toxin: a possible role of component I in the mode of action of the toxin.

C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two separate protein components, designated components I and II, which individually have little activity, but, when mixed and treated with trypsin, exert the potent activity. The present study provides the evidence that component I of the toxin catalyzes the hydrolysis of NAD into nicotinamide and ADP-ribose, whereas component II does not, indicating that component I of C2T has NAD-glycohydrolase activity, which ability is shared with cholera and diphtheria toxins. However, C2T affected neither glycerol production of fat cells nor protein synthesis in cell-free system. Component I of C2T in the presence of [alpha-32P]NAD radiolabeled a protein of Mr 46,000 in the supernatant fractions of mouse tissue homogenates; the protein was abundant in brain, lung and intestine, whereas there was little or none of the protein in muscle. These results indicate that component I can catalyze the covalent attachment of the ADP-ribose moiety of NAD to intracellular protein, which differs from those modified with cholera and diphtheria toxins. The present data, together with previous findings, suggest that the biological activity of C2T is elicited by ADP-ribosylation activity of component I, which is internalized into the cells after binding to the receptor site introduced with the binding of component II to the cell surface membrane.

Carbohydrate storage in man: speculations and some quantitative considerations.

Recent information indicates that the capacity of man to store carbohydrate energy by transformation into fatty acids synthetized de novo is very limited in adipose tissue as well as in liver and intestine. This seems to be in contrast to other species such as the rat where de novo fatty acid synthesis can be induced to a high capacity of glucose removal. This leaves man with a limited capacity to store excess carbohydrate. The remaining possibilities are both the main glycogen stores in liver and in muscle. The latter is by far the largest. The capacity of muscle to assimilate glucose is dependent on its glycogen content that in turn is dependent on previous glycogen depletion to supply energy for muscle contraction. Man might, thus, be uniquely limited in the capacity to dispose of extra carbohydrate in the sedentary state. This might speculatively be thought to be an explanation for a carbohydrate excess syndrome in the sedentary state that may well increase the risk for obesity, hyperinsulinemia, and diabetes mellitus. The logical treatment for such a syndrome then is either a decreased intake of energy as carbohydrate or an increased disposal of carbohydrate energy by exercise. Exercise has, indeed, been shown to have such effects both after physical training programs and, perhaps more pertinent to the question, during a few days after a single exercise bout that has consumed a large amount of muscle glycogen.

Glyceroneogenesis in rat and guinea pig adipose tissue during the fed and fasted states.

While both pyruvate and lactate are good substrates for glyceride-glycerol synthesis in isolated adipocytes from fed rats and guinea pigs, neither alanine nor serine appear to support glyceroneogenesis. Fasting increases the proportion of radioactive pyruvate or lactate incorporated into glyceride-glycerol and reciprocally decreases the proportion incorporated into fatty acids. However, the total incorporation of radioactivity into triacylglycerol is considerably lower in isolated adipocytes from fasted than from fed animals. Addition of glucose to the incubation medium promotes the incorporation of radioactive lactate into both fatty acids and glyceride-glycerol by adipocytes from fasted as well as fed animals. The concentration of alpha-glycerolphosphate is considerably higher in adipose tissue of fed than fasted animals. In general, these results support the presence of a glyceroneogenic pathway in rat and guinea pig adipose tissue. However, it would appear that the physiologic significance of this pathway is less important in the fasted than the fed state, where it may play some role in the esterification of intracellular fatty acids.

Regional differences in the control of lipolysis in human adipose tissue.

Omental fat cells were 30% smaller than those in subcutaneous regions. In omental fat cells with a mean diameter of 95 mu, the basal cAMP concentration was 50% lower, but the basal rate of glycerol release was three times as rapid as in subcutaneous (epigastric) fat cells of identical size. Added at maximal effective concentration, noradrenaline increased the level of cAMP and the rate of glycerol release more markedly in the omental than in the subcutaneous adipocytes, whereas the response to isopropyl noradrenaline was similar. Before starvation the lipolytic effects of noradrenaline and isopropyl noradrenaline, respectively, were identical in the two regions of subcutaneous adipose tissue investigated (femoral and hypogastric). The findings were well related to the tissue levels of cAMP induced by the two agents. During starvation noradrenaline and isopropyl noradrenaline increased the cAMP level and the rate of lipolysis in fat cells obtained from the hypogastric region, whereas noradrenaline decreased these parameters in femoral adipocytes. Starvation was associated with a more prominent inhibitory effect of phenylephrine on basal and isopropyl-noradrenaline-induced lipolysis in femoral than in hypogastric adipose tissue. In conclusion, differences exist between different regions of adipose tissue in their lipolytic responsiveness to noradrenaline, which seems related to the balance between alpha- and beta-adrenergic receptor response.

Effect of dietary fats on ovine adipose tissue metabolism.

The effects of different dietary fats on ovine adipose tissue metabolism have been investigated. Six-month old sheep were fed for 6 weeks a control diet or diets supplemented with either tallow or a mixture of sunflower seed oil and soybean oil, treated to protect the fats from hydrolysis and hydrogenation in the rumen, or with maize oil. The rates of fatty acid, glyceride glycerol, and CO2 formation were measured in perirenal and subcutaneous adipose tissue slices by following the incorporation of either 14C from labeled acetate or glucose, or 3H from tritiated water into the appropriate product. Feeding protected tallow or maize oil but not protected sunflower seed oil plus soybean oil resulted in reduced rates of fatty acid biosynthesis in both perirenal and subcutaneous adipose tissue slices and CO2 formation in perirenal adipose tissue. Feeding the fat-supplemented diets had no effect on the rate of glyceride glycerol formation. The fat-supplemented diets also resulted in reduced activities of various enzymes, thought to be involved in lipogenesis, measured in 105,000 x g supernatant fractions from adipose tissue homogenates. The results suggested that ovine adipose tissue lipogenesis is sensitive to both the amount and the nature of dietary fat.

Effect of lactation on lipolysis in rat adipose tissue.

The concentrations of nonesterified fatty acids and glycerol in rat parametrial adipose tissue increased at peak lactation. Adipose tissue from lactating rats showed higher rates of release of nonesterified fatty acids and glycerol when incubated in vitro than did tissue from nonlactating rats, but there was a substantial increase in the esterification of fatty acids during involution. These results support earlier evidence that fat reserves were mobilized during lactation.

PIP: The effect of lactation on the content of nonesterified fatty acids (NEFA) in rat adipose tissue was studied in vitro. NEFA content was significantly (P less than .05) increased over unmated control values on Day 14 of lactation, as were glycerol levels on Day 14 and during involution. The addition of epinephrine to the incubation medium stimulated lipolysis. The results demonstrate the increased metabolic activity of adipose tissue during lactation in response to the demands of milk production.

The effects of administering N-(2-benzoyloxyethyl) norfenfluramine to rats on the hepatic synthesis of glycerolipids.

N-(2-Benzoyloxyethyl) norfenfluramine (S-780) was administered to rats by stomach tube at a dose of 50 mg kg-1 of body weight. Livers of the rats which were given an acute dose of the drug synthesized more triacylglycerol, phosphatidylcholine and phosphatidylethanolamine from [1,3-3H]glycerol and [14C]palmitate than did those of control rats. The measurements were made by injecting a mixture of the radioactive precursors into the portal veins of anaesthetized rats and freeze clamping a portion of the liver 1 min later. Diffferent results were obtained after treating rats daily with S-780 for 5 days. Liver slices from these rats synthesized less triacylglycerol and relatively more phosphatidylinositol plus phosphatidylserine from [3H]glycerol than did those of control rats. S-780 treatment depressed the hepatic synthesis of phosphatidylcholine and phosphatidylethanolamine as measured in vivo after intrapotal injection of [14C]palmitate and [3H]glycerol. Chronic treatment with S-780 also depressed food intake and lowered liver weight and body weight of rats fed the 41B diet. The results are discussed in relation to the effects of S-780 on the synthesis of glycerolipids.

[Hormonal control of prostaglandins biosynthesis during lipolysis in rat adipose tissue (author's transl)]. / Contrôle hormonal de la biosynthèse des prostaglandines au cours de la lipolyse dans le tissu adipeux de rat

Lipolysis in rat fat pads was studied by determination of free fatty acid and glycerol production in various experimental conditions (absence or presence of glucose, adrenaline and insulin). These results were compared to the accumulation of endogenous prostaglandins E2 and F2 alpha during lipolysis. In the absence of glucose the prostaglandin production followed the adrenaline-ininduced fluctuations in released free fatty acids both in the presence or absence of insulin. In the presence of glucose and insulin, a drop in prostaglandin accumulation was observed whereas free fatty acid production was strongly stimulated. These results suggest either that free fatty acid composition is modified influencing the acitvity of prostaglandin synthetase, or that there exists a specific mechanism controlling prostaglandin synthesis.

[Enzymatic hydrolysis of fat in the presence of Lamblia duodenalis (in vitro)]. / Izuchenie fermentativnogo gidroliza zhira v prisutstvii Lamblia duodenalis (in vitro).

The influence of Lamblia on the hydrolysis of fat by lipase was studied in vitro. The hydrolysis rate of fat in the presence of live Lamblia and without them was determined colorimetrically by the quantity of the formed glycerine. In addition, the kinetics of this reaction was studied by the method of compensating potentiometry by neutralization of fat acids with alkali. The intact organisms were found to cause an inhibition of fermentative hydrolysis of fat. The importance of this fact from the point of view of interaction in the host-parasite system is discussed.