Glycerol Production Rate Research Articles

Glycerol production in subcutaneous adipose tissue in lean and obese humans.

To estimate the regional subcutaneous glycerol production rate in normal and obese humans, the venous arterialized plasma glycerol, interstitial glycerol in the subcutaneous adipose tissue together with adipose tissue blood flow (ATBF, ml/100 g.min) were measured in the postabsorptive state and for 2 h after ingestion of 100 g of oral glucose. Eight lean and eight obese men with normal oral glucose tolerance tests were investigated with the subcutaneous microdialysis technique and 133Xe clearance. In the postabsorptive state, the interstitial glycerol concentrations in lean and obese subjects were 170 +/- 21 vs. 282 +/- 28 microM (P less than 0.01) and 156 +/- 23 vs. 225 +/- 12 microM (P less than 0.05) in the abdominal and femoral subcutaneous adipose tissue, respectively. The corresponding arterial glycerol levels were 54 +/- 4 vs. 75 +/- 14 microM (NS). Abdominal ATBF was greater in lean subjects (3.2 +/- 0.6 vs. 1.6 +/- 0.3; P less than 0.05), whereas femoral ATBF was similar in both groups (2.7 +/- 0.4 vs. 2.4 +/- 0.7). Estimated mean local glycerol release (mumol/100 g.min) was similar in the lean and obese group (0.16 +/- 0.03 vs. 0.20 +/- 0.05 and 0.18 +/- 0.02 vs. 0.17 +/- 0.04) in the abdominal and femoral site, respectively. We conclude that glycerol production from the subcutaneous tissue is increased in obesity, irrespective of adipose tissue distribution. This enhancement is due to the increased adipose tissue mass.

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.

Strategies of freeze avoidance in larvae of the goldenrod gall moth, Epiblema scudderiana: Winter profiles of a natural population

Larvae of the goldenrod gall moth, Epiblema scudderiana (Clemens) utilize a freeze-avoidance strategy for winter survival. Cold-hardiness adaptations of an outdoor population of the species were profiled over the 1984–1985 winter. Over the autumn months supercooling points of the larvae dropped from −13.9±2.3°C to −37.8±2.8°C (the lowest winter temperature recorded was −26°C), water content of the larvae decreased from 57.2±1.2 to 24.8±1.6% of fresh weight, and glycerol content of the larvae rose to an average of 2030 μmol/g wet weight or 18.7% of fresh weight. All parameters stabilized over the mid-winter months. Glycerol production was largely accounted for by the loss of stored glycogen while lipid and protein reserves remained nearly constant over the winter months. Supercooling-point depression and glycerol systhesis both appeared to be initiated after the first overnight exposures to subzero temperatures. Highest rates of glycerol production, about 60 μmol g −1 d −1, were achieved with mean daily temperatures of about 0°C and subzero nights. Glycerol content was rapidly cleared in the spring but only 20% of the resulting carbon was restored as glycogen.

Glycerol production by immobilised cells of Pichia farinosa

Cells ofPichia farinosa were immobilised in calcium alginate and K-Carrageenan and their ability to produce glycerol from glucose under aerobic conditions with acidic as well as alkaline pH was investigated. An average glycerol production rate of 0.07 g/l.h was obtained with immobilised cells (IMC) in shake flasks. Continuous glycerol production in a fluidised bed reactor (FBR) under steady state operation gave a glycerol concentration of 13.5 g/l in the product stream.

Studies on the involvement of lipolytic enzymes in endogenous lipolysis of the isolated rat heart

Rat hearts were depleted in vivo from both the heparin-releasable lipoprotein lipase and heparin-resistant tissue neutral triacylglycerol lipase activity by treatment of the animals with cycloheximide (2 mg/kg body weight), intraperitoneally injected 2.5 and 5 h prior to perfusion. The tissue acid lipase, mono- and diacylglycerol lipase activities were not affected by cycloheximide-induced inhibition of protein synthesis. Myocardial basal and glucagon-stimulated lipolysis, determined by the rate of glycerol production and release from the isolated hearts, was not significantly different in control and cycloheximide-treated rats. Tissue triacylglycerols were recovered with the highest relative specific distribution in the lysosomal fraction isolated from heart homogenates. Upon prolongation of the perfusion-duration the relative specific distribution of triacylglycerols in the lysosomal fraction decreased. In addition, the specific lysosomal triacylglycerol content (μg/mg protein) dropped significantly, indicating an important role of lysosomes in myocardial triacylglycerol turnover. Our data strongly suggest that the heparin-resistant neutral triacylglycerol lipase activity may not be the only determinant of endogenous lipolysis in the isolated rat heart and indicate that lipolysis may additionally be mediated by the lysosomal, acid lipase in concert with the microsomal mono-and diacylglycerol lipase.

Evidence for a substrate regulation of triglyceride lipolysis in human skeletal muscle.

Glycerol release from the human forearm which is generally used as a semiquantitative index of intramuscular lipolysis was studied under different hormonal influence and substrate supply in healthy volunteers and juvenile diabetics using the forearm technique. Acute insulin deficiency in juvenile diabetics failed stimulating the rate of muscular lipolysis since the rates of glycerol release in normals and diabetics were the same. In addition, in normal volunteers high physiological levels of insulin caused by an intraarterial infusion of the hormone exhibited no effect on the glycerol release from deep forearm tissue. Similarly, an intraarterial infusion of metaproterenol did not accelerate muscular glycerol release in normal man. However, in juvenile diabetics in acute insulin deficiency the same dose of the catecholamine increased the rate of muscular glycerol production. Elevated substrate supply during intravenous infusion of glucose or fructose yielded increased uptake of glucose and fructose into the deep forearm tissue and thereby promptly blocked muscular glycerol release in normal volunteers and in juvenile diabetics. These findings suggest that the rate of lipolysis in muscle tissue is not primarily under the control of hormones but rather by substrate supply.

Glycerol‐3‐phosphate dehydrogenase (EC 1.1.1.8) from Dunaliella tertiolecta. I. Purification and kinetic properties

A rapid purification procedure for glycerol‐3‐phosphate dehydrogenase from Dunaliella tertiolecta (strain 19‐6 of the algal collection of the Univ. of Göttingen), the initial enzyme in the glycerol cycle, has been developed on the basis of affinity chromatography on Blue Sepharose and subsequent desalting by Sephadex G‐50. The achieved purification was 126‐fold. The pH optimum of dihydroxyacetone phosphate reduction is 7, that of glycerol‐3‐phosphate oxidation is about 9. The in vitro enzymatic activity obtained from cell extracts is higher than the required activity for the observed glycerol production rates under osmotic stress in vivo.

Effects of hormones on the rate of the triacylglycerol/fatty acid substrate cycle in adipocytes and epididymal fat pads

Brian Brooks, Jonathan R.S. Arch + and Eric A. Newsholme* Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU and + Beecham Pharmaceuticals Research Division, Biosciences Research Centre, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey KT18 5XQ, England Received 12 July 1982 Hormone Triacylglycerol/ fatty acid substrate cycle Substrate cycling Noradrenaline Glucagon Triacylglycerolsynthesis 1. INTRODUCTION It has been suggested by some workers that sub- strate ('futile') cycles are either of little of no sig- nificance in metabolism [1] whereas others suggest that they are of considerable importance in in- creasing sensitivity in metabolic control [2-4]. One prediction of the hypothesis that cycling increases sensitivity is that the rate will vary from one con- dition to another and that some hormones may specifically increase the cycling rate. Evidence for the existence of the triacylglycerol/fatty acid cycle in adipose tissue was presented by Steinberg [5] who compared the rates of fatty acid and glycerol production by isolated epididymal fat pads. This method has been used to investigate the effect of some hormones on the rate of the triacylglycerol/ fatty acid cycle in isolated adipocytes of the rat. In addition, a radiochemical method that depends upon the incorporation of tritium from tritiated water into the glycerol and fatty acid moieties of triacylglycerol has been developed for the mea- surement of the rate of this cycle in epididymal fat pads of the rat. The effects of some hormones have been investigated using both techniques and the results are reported and discussed here. 2. MATERIALS AND METHODS Male Wistar rats were obtained from OLAC (1976) Ltd. (Blackthorn, Bicester, Oxon OX60TP). * To whom correspondence should be addressed Chemicals, biochemicals and enzymes were ob- tained from sources given in [6] except that crys- talline glucagon, ACTH and TSH were obtained from Sigma Chemical Co. (Poole, Dorset) and radiochemicals from The Radiochemical Centre (Amersham HP7 9LL). Rats were killed by cervical dislocation and the epididymal fat pads were removed. Only the thin- ner distal portion of the pad was used for incuba- tion and up to 200 mg fresh tissue was incubated in 1.5 ml Krebs-Ringer bicarbonate buffer con- taining 1 mM glucose and 4% (w/v) defatted al- bumin [6]. Fat cells were prepared as in [7] and in- cubated in 1.3 ml Krebs-Ringer buffer as in [6]. The incubation was terminated by addition of 300/tl 2 M H2SO4 (which does not precipitate al- bumin); 200 ttl was taken for assay of fatty acid and 1 ml for assay of glycerol. For the latter, the cells and albumin were precipitated by addition of trichloroacetic acid (200/d, 40% (w/v)). The pre- cipitate was removed by centrifugation and univer- sal indicator (15/~1) was added to the supernatant and neutralised by addition of 7 M KOH from a microsyringe. The concentrations of glycerol and fatty acids were determined as in [8,9]. The rate of cycling was calculated as follows: cycling -- [(3 x rate of glycerol production)

Lipolytic and circulatory responses to noradrenaline infusion in hypothyroid subjects before and during thyroxine substitution

The hypothyroid state in humans is associated with a diminished lipolytic response to noradrenaline in adipose tissue in vitro . In the present study we have investigated the situation in vivo in order to see if such a change in receptor response could be demonstrated in adipose tissue and in the circulatory system. The change in glycerol production rate, induced by an infusion of noradrenaline, was used as an index of adipose tissue adrenergic responsiveness. The results showed that the lipolytic response was decreased by about 50% in the hypothyroid state and that it was normalized when the substitution dose had been increased to 0.10–0.15 mg/day thyroxine. The circulatory response was monitored by measurements of blood pressure and pulse rate. The resting diastolic pressure was transiently lowered by substitution. Similarly the rise in systolic blood pressure induced by l-noradrenaline was transiently increased by substitution. Thus no clear-cut change in receptor response with substitution could be demonstrated by measuring BP and pulse rate only. This result could be due to the fact that the system is more complex than the adipose tissue. The finding of a reduced adrenergic receptor response in vivo in the adipose tissue is in accordance and extends earlier findings in vitro .

Studies on glycerol kinase and its role in ATP synthesis in Trypanosoma brucei.

Glycerol kinase of Trypanosoma brucei has been shown to be capable of catalysing sn-glycerol-3-phosphate dependent ADP phosphorylation for ATP generation. The rate of this reaction (Vr) is sufficient to account for the observed rate of glycerol production from anaerobic glucose metabolism by intact cells and to account for net ATP synthesis. Glycerol kinase has been purified by preparing a post-nuclear, particulate fraction and solubilizing the enzyme with 0.5% (w/v) Triton X-100. This treatment results in a 3.5-fold increase in total activity, demonstrating the latent nature of particulate glycerol kinase, and an overall 10-fold increase in specific activity in the soluble fraction. The ratio of the velocities of the forward (Vf) reverse (Vr) reactions of this enzyme is altered from 21 to 170 upon solubilization. The Michaelis constants for the solubilized enzyme are KmADP = 0.12 +/- 0.04 mM, KmG-3-P = 5.12 +/- 1.47 mM, Kmglycerol = 0.12 +/- 0.05 and KmATP = 0.19 +/- 0.04 mM. Endogenous hexokinase acts as an ATP trap favouring ATP synthesis sn-glycerol-3-phosphate and ADP. This can be demonstrated in reconstituted systems using trypanosome glycerol kinase and varying hexokinase activities. Mass action inhibition of ATP synthesis by glycerol is more marked with lower hexokinase activities. High glycerol kinase activity (> 0.5 mumol/min/mg protein) has been found in the T. brucei complex of trypanosomes that produce glycerol anaerobically whereas only low activities (less than or equal to 0.03 mumol/min/mg protein) are present in Trypanosoma cruzi, Trypanosoma lewisi and Crithidia fasciculata, organisms that do not produce glycerol. Trypanosoma congolense has a glycerol kinase activity of 0.17 mumol/min/mg protein and shows poorer ATP synthesis from anaerobic glucose metabolism than organisms of the T. brucei complex.

Influence of experimental hyperthyroidism on skeletal muscle metabolism in the rat.

In this study hind-limb perfusion was used to investigate the influence of thyroid hormones on some metabolic parameters in the skeletal muscle of the rat. Daily injection of 20 microng L-thyroxine (T4) per 100 g b. w. for a week caused a 25% increase in oxygen consumption. Further enlargement of the T4 dose had little additive effect. In the dose range 20--80 microng T4/100g b.w., no important changes occurred in lactate production or glucose consumption. Only at the highest T4 dose did the glucose consumption increase significantly. The most profound effect of T4 was on lipolysis. A daily dose of 20 microng T4/100 g b. w. gave a doubling of glycerol production rate, the maximum occuring at a dose of 40 microng T4/100 g b. w. Inactivation of the nervous system was without influence on the T4-induced increase in oxygen consumption. However, the T4-induced elevation of lipolysis disappeared after abolition of the nervous activity. This raises the possibility that the T4 effect on lipolysis in skeletal muscle is a potentiation of catecholamine effects. The T4-induced oxygen consumption increase might be dependent not on the lipolytic process but rather on other energy-consuming cell processes.

Studies on the mechanisms of epinephrine stimulation of lypolysis

The time course for epinephrine stimulation of lypolysis, cyclic AMP accumulation and activation of protein kinase was studied in adipose tissue from hypophysectomized rats. Triglyceride breakdown, as assessed by glycerol release, increased rapidly in response to epinephrine, maintained a constant rate as long as the hormone was present, and decreased rapidly to basal values when the hormone was removed. Cyclic AMP accumulation was transient peaking within 3 min of exposure to epinephrine and then declining to levels indistinguishable from basal by 9 min. Protein kinase activity in extracts also peaked at 3 min and thereafter declined to a level approximately 25% greater than resting activity. Peak levels of cyclic AMP, steady state levels of protein kinase activity and the rate of glycerol production were all related in a dose dependent manner to the concentration of epinephrine. These observations suggest that the spike in cyclic AMP levels may be necessary to trigger the activation of lipolysis, but was not sufficient to sustain an accelerated rate of tryglyceride breakdown. Continued activation of protein kinase, however, may be essential to sustained lipolysis.

Changes in the Adrenergic Control and the Rate of Lipolysis of Isolated Human Adipose Tissue during Fasting and after Re‐feeding

The influence of changes in nutritional state on lipid mobilization has been investigated, using subcutaneous fat portions removed from 8 obese patients submitted to fasting for one week. The maximal rise in the tissue level of cyclic AMP (cAMP) and the rate of glycerol release were determined when adipose tissue was exposed to noradrenaline (6 X 10(-6) mol/1), isopropylnoradrenaline (6 X 10 (-6) mol/1) or none of the agents (basal medium). Fasting resulted in significant increases in the tissue level of cAMP and the rate of lipolysis in adipose tissue unexposed to lipolytic agents. Noradrenaline decreased the level of cAMP, in contrast to isopropylnoradrenaline, and significantly inhibited the rate of lipolysis during fasting. These noradrenaline effects were abolished by the simultaneous presence of phentolamine (13 mmol/1) in the incubation medium. Re-feeding with Meritene for one day resulted in a diminished rate of basal lipolysis, whereas the cAMP level was unaffected. The response of the cAMP accumulation to isopropylnoradrenaline was further augmented by re-feeding. Noradrenaline produced a significant rise in the level of cAMP and significantly stimulated the rate of glycerol production. It is concluded that the nutritional changes are of significance for the adrenergic regulation of lipolysis as indicated by the response of cAMP to the catecholamines. In pharmacological terms, fasting for one week resulted in increased alpha-as well as beta-adrenergic responsiveness. Increased basal lipolysis during fasting may be related to an increased level of cAMP or a direct activation of lipase.

Selective loss of adipose cell responsiveness to glucagon with growth in the rat

In isolated fat cells, the same maximal rate of glycerol production can be induced by epinephrine or ACTH, alone or in combination with each other or with glucagon. With fat cells from rats weighing 150-175 g, the maximal rate of lipolysis attained with glucagon was 75-80% of that produced by epinephrine or ACTH, and with increasing size of the donor rat, the magnitude of the effect of glucagon relative to that of the other hormones declined markedly. In particulate preparations from fat cells of rats weighing 100-125 g, the maximal effect of glucagon on adenyl cyclase activity was about 60% of that of epinephrine, and was significantly less (30%) in preparations from 350-400 g rats. These data are consistent with the hypothesis that with growth of the rat there is a selective decline in the number of glucagon receptors relative to those for epinephrine or ACTH in the fat cell membrane.

Effects of Lipolytic and Antilipolytic Agents on Cyclic 3',5'-Adenosine Monophosphate in Fat Cells

Abstract Isolated fat cells were incubated in Krebs-Ringer phosphate medium. Within 1 min after the addition of 1.1 µm epinephrine, the cyclic AMP content of the cells was elevated. It continued to rise, reaching a peak in 4 to 7 min, and fell thereafter, being only slightly above the control levels after 10 to 15 min. A maximal rate of glycerol production was established within 1 to 2 min, well before the peak in cyclic AMP concentration, and was maintained unchanged while the cyclic AMP level continued to rise and then fell. Both in magnitude and time course, the effect of adrenocorticotropin (ACTH), 0.2 unit per ml, on cyclic AMP levels and on lipolysis was exactly like that of epinephrine (or of both hormones together). Addition of more epinephrine or of ACTH at several times during 1 hour of incubation with 1.1 µm epinephrine did not change cyclic AMP levels. The effects of epinephrine on cyclic AMP levels and on glycerol production were unaltered by the presence in the medium of 4 mm glucose. When propranolol (or insulin) was added several times after epinephrine-simulated lipolysis had begun, the rate of glycerol production fell to zero within 1 to 2 min. In the same period, the cyclic AMP concentration fell to a basal level; i.e. the rate of decrease was much greater than that observed after 4 to 7 min of incubation with epinephrine alone. Furthermore, after incubation of cells for 20 min with epinephrine, the addition of 0.8 mm theophylline produced an 8-fold rise in cyclic AMP content measured 5 min later. This increment was, however, only a fraction of that observed during the first 5 min after addition of epinephrine in the presence of theophylline. Since propranolol inhibits the action of epinephrine but does not alter basal adenyl cyclase activity, and, since 0.8 mm theophylline alone did not alter cyclic AMP levels, it may be concluded that adenyl cyclase was still epinephrine-stimulated during the time when cyclic AMP levels were falling. Whether the rate of formation of cyclic AMP during this period was equal to that during the first minutes after addition of the hormone cannot be established. Attempts to show directly alterations in phosphodiesterase activity during incubation of cells with epinephrine have been unsuccessful. When the effect of epinephrine was terminated after 4 min by the addition of propranolol, introduction of ACTH at several times during the following 20 min caused an immediate resumption of glycerol production at a rate equal to that observed with the initial addition of epinephrine. Cyclic AMP levels, however, rose little if at all in 4 to 5 min after addition of ACTH. The ability of cells to elevate cyclic AMP levels a second time in response to hormonal stimulation was restored when cells that had been incubated for 20 min were washed and suspended in fresh medium. There appears to be something accumulated in the medium during incubation of cells with epinephrine that interferes with the effectiveness of the hormone in causing accumulation of cyclic AMP in fresh cells. Whether this material has its effect on the rate of formation or of degradation of cyclic AMP cannot be decided.

Glycerol metabolism in rabbits.

The endogenous rate of glycerol production in rabbits was measured by several techniques: constant infusion of 1,3-14C-glycerol or 2-3H-glycerol or unlabeled glycerol; single injection of 1,3-14C-glycerol or 2-3H-glycerol or unlabeled glycerol. The rate was 5.5–11.6 μmoles/kg per minute (9 rabbits). The mean fractional turnover rate was 0.0585 ± 0.0052. During infusion of noradrenaline together with 3H-glycerol, the fractional turnover rate was no different from that in the absence of noradrenaline. The maximum utilization rate of glycerol was 28.1 ± 1.40 μmoles/kg per minute. The glycerol space was 58.1% of body weight. The relationship of glycerol concentration to rate of glycerol utilization in the intact rabbit suggests the existence of an enzyme with a KM for glycerol of 0.33 × 10−3 M; the glycerol kinase of rabbit liver was found to have a KM for glycerol of 0.29 × 10−3 M. This enzyme could account for the disappearance of glycerol in the intact animal except that its Vmax is only 4% of that expected. Possible reasons for this are discussed. A glycerol dehydrogenase with a Vmax similar to that of the glycerol kinase also exists in rabbit liver; its KM for glycerol is so high (0.5 M) that it is unlikely to play a significant role in glycerol metabolism in the normal rabbit.

The action of cyclic 3′,5′-adenosine monophosphate on lipolysis in rat adipose tissue

1. 1. Stimulation of lipolysis in fat cells or intact adipose tissue by cyclic 3',5'-AMP, 1 mM, was readily demonstrated in simple phosphate-saline (all Na +) medium. Adenine, 1 mM, also to a lesser extent stimulated lipolysis in this medium but unlike cyclic 3',5'-AMP its effect was not diminished by the presence of K + in the incubation medium. Adenosine, 5'-AMP, ADP, or ATP did not enhance lipolysis. 2. 2. In phosphate-saline medium with K + (7 mequiv/1) cyclic 3',5'-AMP stimulated lipolysis when the basal rate was low (tissue from fasted-refed rats, tissues preincubated with glucose and insulin) and inhibited lipolysis when the basal rate was high (tissues from fasted rats, tissues incubated with epinephrine). 3. 3. In Krebs-Ringer phosphate medium cyclic 3',5'-AMP alone had no effect on production of glycerol by fat cells and decreased the elevated rate of glycerol production associated with the presence of theophylline. In Krebs-Ringer phosphate medium without Ca 2+ and Mg 2+, cyclic 3',5'-AMP alone stimulated glycerol production and did not decrease glycerol production when theophylline was present. 4. 4. EDTA (5 mM) increased glycerol production in medium without Ca 2+ and Mg 2+. Alone, it had no effect in medium with Ca 2+ and Mg 2+ but inhibited the effect of epinephrine. Stimulation of lipolysis by EDTA (no Ca 2+ or Mg 2+) was much greater in all Na + medium than it was in the presence of K +. The composition of free fatty acids released into the medium was similar whether lipolysis was stimulated by epinephrine, cyclic 3',5'-AMP or EDTA. 5. 5. Stimulation of lipolysis by cyclic 3',5'-AMP (all Na + medium) was not observed in the absence of phosphate which itself in high concentration (>20 mM) increased release of free fatty acids.