Lipid composition of the fat body and haemolymph and its relation to lipid release in Oncopeltus fasciatus

Lipid composition of the fat body and haemolymph of male milkweed bug, Oncopeltus fasciatus, was determined. Triglycerides were the predominant lipids of the fat body while diglycerides accounted for the major lipid in the haemolymph. Sterols, sterol esters, and non-esterified fatty acids were present in both fat body and haemolymph besides triglycerides and diglycerides. Only traces of monoglycerides were detected.Gas chromatographic analysis of the fatty acids revealed a difference in the fatty acid composition between fat body and haemolymph glycerides and sterol esters. Oleate and linoleate were the predominant unsaturated fatty acids in both fat body and haemolymph lipids and in the milkweed seeds as well.When fat body was labelled in vivo and in vitro with ¹⁴C-palmitate, the fatty acid was incorporated largely into the triglycerides. When the prelabelled fat body was incubated with a medium containing haemolymph the fat body released lipids mainly as diglycerides. Some radioactivity was observed in the triglycerides and non-esterified fatty acids also.Electrophoretic analysis of the incubation medium containing the haemolymph revealed that the released lipids were bound to three haemolymph lipoprotein bands. Lipid mobilization, release, and transport in Oncopeltus are discussed in relation to studies on other insects.     

Studies on the respiratory metabolism of isolated human adipose cells

1. Some metabolic characteristics of fat cells isolated from 50 patients were examined. 2. The respiratory activity of human fat cells was of the same order of magnitude as cells obtained from the rat, whether the comparison was based on the molar triglyceride content of cells or the nitrogen content of tissue. 3. There was no difference in the respiratory metabolism of fat cells isolated from three separate age groups: 20-40, 40-60 and 60-80 years. 4. The respiratory activity of human adipose cells was partially inhibited by malonate (0.1m), arsenite (1mm) and iodoacetate (1mm). 5. Oxygen consumption by human adipose cells could not be stimulated by adding glucose or glucose plus palmitic acid to the medium. However, stimulation of oxygen uptake with these substrates was observed after the tissue had been preincubated for 5hr. in a bicarbonate buffer. 6. After the tissue had been preincubated for 5hr., there was a linear relationship between the oxygen uptake by isolated cells and the amount of oleic acid added to the incubation medium. Stimulation of oxygen uptake by oleic acid (0.95mumole/ml.) was dependent on the presence of glucose (5.6mm) in the incubation medium. 7. Of the total [1-(14)C]palmitic acid metabolized by human adipose cells, over 99.8% of the radioactivity was recovered in the neutral-lipid fraction and less than 0.2% in carbon dioxide. Under conditions where oxygen uptake is stimulated by [(14)C]-palmitic acid, the radioactivity of the 1,2-diglyceride pool increased 20-fold.     

Release of free fatty acids from Ehrlich ascites tumor cells

Ehrlich ascites tumor cells release free fatty acids (FFA) during in vitro incubation in media that contain albumin. The released FFA are derived by lipolysis from endogenous lipid esters. Addition of glucose to the incubation medium greatly decreases the quantity of fatty acid released by the cells. Cyanide, which inhibits endogenous lipid oxidation but not lipolysis, increases the quantity of fatty acid released to media containing albumin and causes free fatty acid to accumulate in the cells in the absence of exogenous albumin. The release of fatty acid, either preformed or derived by lipolysis during prolonged incubations, occurs under conditions of net fatty acid uptake from the incubation medium. Net release of fatty acid from the cell occurs only when fatty acid-extracted albumin is present in the extracellular medium; extrapolation of the data suggests that net release will not occur under physiological conditions. It is postulated that free fatty acid uptake and release are independent processes, the direction of net fatty acid movement being determined by the relationship between cellular free fatty acid concentration (regulating efflux) and the molar ratio of free fatty acid to albumin in the extracellular medium (regulating uptake).     

Effects of various non-esterified fatty acids on the transfer of cholesteryl esters from HDL to LDL induced by the cholesteryl ester transfer protein

The effects of saturated, monounsaturated and polyunsaturated non-esterified fatty acids on the rate of transfer of radiolabeled cholesteryl esters from high density lipoproteins (HDL) to low density lipoproteins (LDL), induced by the cholesteryl ester transfer protein (CETP), have been studied. Human high-density lipoproteins-subfraction 3 (HDL3) containing radiolabeled cholesteryl esters were incubated with LDL at 37 degrees C with or without CETP and in the absence or in the presence of non-esterified fatty acids. Less than 6% of the total radioactivity was recovered in the LDL fraction after incubation of HDL3, and LDL for 3 h at 37 degrees C in the absence of CETP, regardless of whether or not non-esterified fatty acids were added. The addition of CETP to the incubation mixture induced a time-dependent redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. Non-esterified fatty acids were found to alter the rate of transfer of cholesteryl esters induced by CETP. While short chain saturated non-esterified fatty acids (caprylic and capric acids) had no effect on the rate of transfer of cholesteryl esters, the medium and long chain ones (lauric, myristic, palmitic and stearic acids) significantly increased the CETP-mediated transfers from HDL3 to LDL. At low concentrations, unsaturated fatty acids also stimulated the CETP-mediated redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. As the concentration of either oleic, linoleic or arachidonic acids increased to higher levels, a significant proportion of fatty acids remained unassociated with lipoprotein particles. Under these circumstances the transfer process was inhibited. These results show that non-esterified fatty acids can modulate the CETP-mediated transfer of cholesteryl esters from HDL to LDL and that this effect is dependent on both the length and the degree of unsaturation of their monomeric carbon chain.     

Effects of exogenous growth hormone on milk production and nutrient uptake by muscle and mammary tissues of dairy cows in mid-lactation

Responses to exogenous growth hormone were measured in lactating dairy cows surgically prepared to allow measurement of nutrient exchanges across mammary and hind-limb muscle tissues. Cows were injected daily with either saline or growth hormone, at a dose of 0.1 mg/kg liveweight, over periods of 6 days. During administration of growth hormone milk yield, milk fat content and yields of milk fat protein and lactose increased. Arterial plasma concentrations of glucose and non-esterified fatty acids were increased, uptake of glucose by leg muscle tissue decreased, lactate release from leg muscle tended to increase, mammary uptake of non-esterified fatty acids increased, blood flow to leg muscle tended to increase and blood flow to mammary tissue increased during injection of growth hormone. The results show that growth hormone affects supply to and utilization of key nutrients by tissues, resulting in the supply to the mammary gland of additional precursors for milk synthesis.     

Short-term effects of exogenous growth hormone: effects on milk production and utilization of nutrients in muscle and mammary tissues of lactating ewes

Exogenous bovine growth hormone at a dose of 0.1 mg kg-1 liveweight increased yields of milk and milk constituents and milk fat content when injected over 5 days into ewes in mid-lactation. These changes in milk production were associated with changes in the supply to, and utilization of, nutrients by leg muscle and mammary tissues. Arterial concentrations of glucose and non-esterified fatty acids increased significantly, concentrations of lactate and 3-hydroxybutyrate tended to increase, and concentrations of triglycerides associated with very low-density lipoproteins decreased significantly. Growth hormone increased mammary uptake of non-esterified fatty acids, decreased mammary uptake of very low-density lipoproteins and tended to reduce the release of lactate from leg muscle. Oxidation of non-esterified fatty acids in the whole body and mammary tissue was increased by growth hormone and there was a tendency for reduction of glucose oxidation in mammary tissues. During injection of growth hormone, blood flow to leg muscle and mammary tissues increased as did the calculated ratio of blood flow; milk yield. These changes in blood flow, together with changes in arterial concentrations and tissue utilizations of key metabolites, were sufficient to account for the synthesis of extra milk and milk constituents.     

Insulin and non-esterified fatty acids. Acute regulators of lipogenesis in perfused rat liver.

Livers from fed rats were perfused with whole rat blood and infused with oleate to maintain constant concentrations of serum non-esterified fatty acids over a wide physiological range. Infusion of insulin opposed the antilipogenic effects of increasing concentrations of serum non-esterified fatty acids. Secretion of very-low-density-lipoprotein triacylglycerols was directly proportional to the concentration of serum non-esterified fatty acids and was increased by insulin. The secretion of newly-synthesized fatty acids in very-low-density-lipoprotein triacylglycerols decreased with increasing concentrations of serum non-esterified fatty acid. Insulin opposed this change. Cholesterol biosynthesis was unaffected by alterations in concentration of serum non-esterified fatty acid but was increased by insulin. Equilibrium concentrations of perfusate lactate and glucose were increased by serum non-esterified fatty acids but steady-state rates of hepatic glucose output and lactate uptake were unchanged. Insulin decreased perfusate glucose concentrations and abolished the increase in its concentration that resulted from increases in non-esterified fatty acid concentrations.     

Effect of lipolytic agents on adenosine and AMP formation by fat cells.

The release and metabolism of adenosine was examined using rat fat cells in which the nucleotide pool has been labeled by incubation with radioactive adenine. The accumulation of adenosine in the medium was near maximal at the start of the incubation and increased only slightly thereafter. Adenosine was rapidly deaminated to inosine and subsequently oxidized to uric acid. In the presence of allopurinol, and inhibitor of xanthine dehydrogenase, hypoxanthine accumulated in the medium as the end-product of adenosine catabolism. Adenosine accumulated in the medium only if fat cells were incubated in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. Even in the presence of this inhibitor there was no acceleration of adenosine release by norepinephrine in the presence of theophylline. However, there was an increase in labeled intracellular AMP accumulation by norepinephrine plus theophylline. The increase in labeled AMP correlated with the final free fatty acid to albumin ratio suggesting that the rise in AMP was related to an accumulation of intracellular free fatty acids. The addition of sodium oleate to the medium mimicked the effect of norepinephrine plus theophylline on the accumulation of labeled AMP. These results indicate that AMP rather than adenosine accumulates in isolated fat cells during incubation with lipolytic agents.     

Influence of ethanol on the metabolism of perfused normal, fatty and cirrhotic rat livers

1. The influence of ethanol on the metabolism of perfused livers from normal rats and rats in various stages of development of dietary cirrhosis was studied. A choline-deficient, low-protein and high-fat diet was used. Results were obtained on oxygen consumption and carbon dioxide production, on glucose release and uptake by the liver and on changes in the concentrations of lactate and pyruvate and of β-hydroxybutyrate and acetoacetate in the perfusion medium. 2. Oxygen consumption and carbon dioxide production were lower in fatty and cirrhotic livers than in normal livers. Ethanol had no effect on the oxygen consumption of any of the various livers. After addition of ethanol to the perfusion medium carbon dioxide production ceased almost completely in normal livers. Only a slight decrease in the carbon dioxide production occurred in fatty and cirrhotic livers. 3. With every type of liver glucose was released from the liver into the perfusion medium during the initial control period. This release continued after the addition of ethanol to the perfusion medium in experiments with normal and fatty livers, whereas with cirrhotic livers a marked uptake of glucose from the medium was found. A simultaneous release of the glycolytic end products lactate and pyruvate into the medium occurred. 4. The production of ketone bodies was equal in normal and early fatty livers (6 weeks on the fat diet). It was smaller in late fatty livers (3–4 months on the fatty diet) and in cirrhotic livers. 5. The lactate/pyruvate concentration ratio in the perfusion medium increased from 11 to 67 with normal livers, from 12 to 16 with early fatty livers, from 13 to 26 with late fatty livers and from 21 to 55 with cirrhotic livers when the livers were perfused with a medium containing ethanol. The β-hydroxybutyrate/acetoacetate concentration ratio increased from 1·2 to 8·4 in normal livers, from 2·0 to 2·8 in early fatty livers, from 1·2 to 2·4 in late fatty livers and from 2·1 to 4·0 in cirrhotic livers when ethanol was added to the medium. 6. The effects of ethanol on liver metabolism during the development of dietary cirrhosis are discussed and related to human fatty liver and cirrhosis during chronic ethanol consumption.     

Glucose metabolism in isolated fat cells: enhanced response of larger adipocytes from older rats to epinephrine and adrenocorticotropin.

The effects of insulin and of two lipolytic hormones (epinephrine and ACTH1) on the rate and pattern of glucose metabolism were compared during incubation of isolated fat cells, obtained from epididymal fat pads of rats of varying age and degrees of adiposity. Glucose metabolism and the intracellular free fatty acid levels were expressed on a per cell basis and in relation to adipocyte size. The data for total glucose metabolism show that, in contrast to the declining insulin effect observed with adipocyte enlargement, the stimulation of glucose uptake and metabolism by these lipolytic hormones was significantly greater in the larger fat cells from the older fatter rats than in the smaller ones from the younger leaner rats. Lipolytic hormones suppressed, whereas insulin enhanced, fatty acid synthesis; moreover the lipolytic hormones stiumlated glucose ce effect of epinephrine on the intracellular free fatty acid levels was greater in the small fat cells than in the large ones; this effect of epinephrine was markedly curtained by the presence of glucose in the incubation medium, making it unlikely that acceleration of glucose metabolism by the lipolytic stimulus was mediated by an elevation of the intracellular free fatty acid level. The present results show a markedly enhanced capacity of the large adipocytes to accelerate glucose metabolism in response to these liplytic hormones. Thus, in contrast to prevailing notions of declining hormonal responsiveness with expanding fat cell size in older and more obese animals, this study documents an instance of increased hormonal response in enlarged adipocytes and points to the need for a more comprehensive reevaluation of the various hormonal effects in adipocytes of different size.     

FAT AND CARBOHYDRATE METABOLISM IN HUMANS—A Study of Nutritional and Hormonal Effects

As an index to the rate of fat utilization in human subjects, the recovery of all radioactive carbon dioxide in the expired air was measured for one hour following intravenous injection of palmitate-1-C¹⁴. In the normal fasted subject, about 10 per cent of the injected dose was recovered, and the proportion was lowered to about 5 per cent by administration of glucose. With prolonged fasting, the recovery of radioactive carbon dioxide did not change, despite a rising concentration of fatty acids in the serum. This was interpreted as due to the development of a balance between increasing mobilization and oxidation and was thought to indicate increasing fatty acid oxidation.In chronic undernutrition and diabetes mellitus there was increased fatty acid oxidation due presumably to adaptation to a chronic increase in fat utilization for energy.Administration of human growth hormone did not increase fat oxidation but prevented the usual inhibition produced by glucose. This was interpreted to mean that growth hormone increases fat utilization only indirectly by inhibiting the usual preferential utilization of glucose over fat.     

Effect of allatectomy on fat body lipid metabolism of the male desert locust during adult development

The effect of allatectomy on the changes in fat body lipid metabolism during adult development of the male desert locust (Schistocerca gregaria) has been investigated. All the operations were carried out 4 days after adult emergence. Allatectomy had no appreciable effect on the specific activity of acetate incorporation into lipid, but since the fat body tissue grows after the operation the absolute capacity of the whole tissue is increased. There is a gradual increase in the conversion of glucose to lipid between 4 and 12 days after emergence in the allatectomized insects. The amount of glucose completely oxidized to carbon dioxide is gradually decreased after the operation.     

Regulation of 3-hydroxybutyrate formation and secretion of very-low-density-lipoprotein triacylglycerol by perfused livers from fed and starved rats.

Acetoacetate was the sole ketone body formed when livers from starved rats were perfused with minimal concentrations of non-esterified fatty acid. Absence of 3-hydroxybutyrate was related to a low substrate potential, which caused a more oxidized redox state and a decreased [ATP]/[ADP] ratio. Only under conditions of comparable non-esterified fatty acid uptake was lipoprotein triacylglycerol production inversely related to ketogenesis.     

Biosynthesis of caffeine in tea callus tissue

1. A study of caffeine biosynthesis has been made by following the incorporation of radioactive carbon dioxide and methionine into the methylated purines produced by tea callus tissue. 2. The uptake of the radioactive labels into nucleic acid and caffeine was followed over a period of approximately 9h. 3. The distribution of the radioactive labels in both nucleic acid and caffeine was determined after incorporation and subsequent incubation of the tissue in a non-radioactive medium. 4. The results of the experiments indicated that the caffeine arose from purines released from the breakdown of nucleic acids rather than that it was formed directly from a purine pool. 5. A metabolic scheme to show the production of caffeine from the nucleotides of the nucleic acid is discussed.     

The control of fatty acid and triglyceride synthesis in rat epididymal adipose tissue. Roles of coenzyme A derivatives, citrate and l-glycerol 3-phosphate

1. Methods are described for the extraction and assay of acetyl-CoA and of total acid-soluble and total acid-insoluble CoA derivatives in rat epididymal adipose tissue. 2. The concentration ranges of the CoA derivatives in fat pads incubated in vitro under various conditions were: total acid-soluble CoA, 0.20-0.59mm; total acid-insoluble CoA, 0.08-0.23mm; acetyl-CoA, 0.03-0.14mm. 3. An investigation was made of some postulated mechanisms of control of fatty acid and triglyceride synthesis in rat epididymal fat pads incubated in vitro. The concentrations of intermediates of possible regulatory significance were measured at various rates of fatty acid and triglyceride synthesis produced by the addition to the incubation medium (Krebs bicarbonate buffer containing glucose) of insulin, adrenaline, albumin, palmitate or acetate. 4. The whole-tissue concentrations of glucose 6-phosphate, l-glycerol 3-phosphate, citrate, acetyl-CoA, total acid-soluble CoA and total acid-insoluble CoA were assayed after 30 or 60min. incubation. The rates of fatty acid and triglyceride synthesis, calculated from the incorporation of [U-(14)C]glucose into fatty acids and glyceride glycerol respectively, and the rates of glucose uptake, lactate plus pyruvate output and glycerol output were measured over a 60min. incubation. 5. The rate of triglyceride synthesis could not be correlated with the concentrations of either l-glycerol 3-phosphate or long-chain fatty acyl-CoA (measured as total acid-insoluble CoA). Factor(s) other than the whole-tissue concentrations of these recognized precursors appear to be involved in the determination of the rate of triglyceride synthesis. 6. No relationship was found between the rate of fatty acid synthesis and the whole-tissue concentrations of the intermediates, citrate or acetyl-CoA, or with the two proposed effectors of acetyl-CoA carboxylase, citrate (as activator) or long-chain fatty acyl-CoA (as inhibitor). The control of fatty acid synthesis appears to reside in additional or alternative factors.     

Uptake and release of glutamate in cerebral-cortex slices from the rat

1. Cerebral-cortex slices from rat brain, loaded with labelled l-glutamate as a result of aerobic incubation with labelled glucose, lost less than 15% of this glutamate on subsequent incubation in the presence of unlabelled glucose and l-glutamate. This indicates that very little exchange occurs between extracellular l-glutamate and glutamate accumulated in the neurons as a result of glucose metabolism. 2. Slices, loaded with labelled l-glutamate as a result of aerobic incubation in a medium containing unlabelled glucose and labelled l-glutamate, lost more than half of this glutamate on subsequent incubation in the presence of unlabelled l-glutamate. This indicates that exchange occurs between extracellular glutamate and glutamate accumulated in brain slices as a result of its uptake from the incubation medium. 3. Evidence was obtained suggesting that only a part of the glutamate, accumulated in brain slices as a result of its uptake from an incubation medium containing both glucose and l-glutamate, entered the neurons; apparently almost all the rest entered the glia. 4. It is concluded that the slices contain a pool of glutamate, derived from glucose and located in the neurons, which is poorly exchangeable with extracellular glutamate, and another pool of glutamate, derived from extracellular glutamate and located in the glia, which is freely exchangeable with extracellular glutamate.     

Relationship between fatty acid and glucose utilization in Ehrlich ascites tumor cells

Glucose greatly increased total free fatty acid (FFA) esterification by Ehrlich ascites tumor cells. However, the FFA concentration of the cells was not altered. Less exogenous FFA was oxidized to CO(2) at any given extracellular FFA:albumin molar ratio when glucose was available, but increasing amounts of radioactive CO(2) were produced as the FFA:albumin molar ratio was raised, even in the presence of glucose. It is suggested that glucose, by providing either energy or an excess of triose acceptor for fatty acid esterification, stimulated FFA uptake only indirectly, by increasing the utilization of FFA subsequent to initial uptake from the medium, i.e., by increasing the turnover rate of the cellular FFA pool. Availability of glucose decreased the oxidation of endogenous lipid radioactivity and the depletion of endogenous lipid ester radioactivity. Most of the radioactivity utilized was derived from phospholipids, and depletion of phospholipid radio-activity was spared when glucose was available. Depletion of cellular total lipid ester also was spared in the presence of glucose. Availability of FFA did not decrease total glucose uptake or its oxidation to CO(2). Glucose utilization by these cells appears not to be regulated by FFA availability in the manner that Randle and coworkers described for muscle.     

Effects of prolonged incubation of isolated fat cells on their response to hormones stimulating lipolysis and glucose metabolism

1. The metabolism of isolated fat cells from parametrial adipose tissue of starved normal rats was studied during 8hr. incubation. 2. There was a three- to eight-fold increase in conversion of glucose into carbon dioxide, fatty acids and glycerol during the fourth to eighth hours of incubation in 4% albumin buffer over that seen during the first 4hr. of incubation. 3. The addition of growth hormone and dexamethasone to fat cells at the start of the incubation period accelerated lipolysis during the first 4hr. of incubation but no further effect was seen during the fourth to eighth hours of incubation. Addition of growth hormone and dexamethasone to fat cells that had been incubated for 4hr. did not accelerate lipolysis during the next 4hr. whether fat cells were incubated with or without glucose. 4. Fat cells incubated for prolonged periods also displayed a reduced sensitivity to the lipolytic action of adrenocorticotrophic hormone. 5. During prolonged incubation there was no damage to the cells as judged by the retention of two soluble cytoplasmic enzymes, lactate dehydrogenase and malate dehydrogenase, within the cells.     

Effect of glucose utilization inhibitor isolated from the serum and urine of patients with chronic renal failure on the free fatty acid metabolism in rat kidney cortex slices.

The authors studied interference by the inhibitor of glucose utilization with free fatty acid (FFA) metabolism in rat kidney cortex slices. They found that FFA uptake by the kidneys, their incorporation into triglycerides (TG) and oxidation to 14CO2 were lower when the inhibitor was present in the incubation medium. The findings show that the inhibitor, as well as inhibiting glucose utilization in various tissues, interferes directly with fat metabolism.     

Measurement of flow of carbon atoms from glucose and glycogen glucose to glyceride glycerol and glycerol in rat heart and epididymal adipose tissue: Effects of insulin, adrenaline and alloxan-diabetes

1. Flow of carbon atoms from glucose and glycogen glucose to glyceride glycerol, glyceride fatty acids and glycerol was calculated in the perfused rat heart and incubated epididymal adipose tissue from the incorporation of (14)C from [U-(14)C]-glucose (into glyceride glycerol, glyceride fatty acids and glycerol in the medium), and from measurements of the specific activity of l-glycerol 3-phosphate, and the effects of insulin, adrenaline and alloxan-diabetes were studied. Measurements were also made of the uptake of glucose and the outputs of lactate, pyruvate and glycerol. 2. New methods are described for the measurement of radioactivity in small amounts of metabolites (glycerol, glucose 6-phosphate and fructose 6-phosphate and l-glycerol 3-phosphate) in which use has been made of alterations in charge induced by enzymic conversions to effect resolution by ion-exchange chromatography. 3. In hearts the specific activity of l-glycerol 3-phosphate was less than that of glucose in the medium but similar to that of lactate released during perfusion. Because repeated measurements of the specific activity of l-glycerol 3-phosphate was impracticable, the specific activity of lactate has been used as an indirect measurement of glycerol phosphate specific activity. 4. In fat pads, specific activity of lactate was the same as that of glucose in the medium and thus the specific activity of l-glycerol 3-phosphate was taken to be the same as that of medium glucose. 5. In hearts from alloxan-diabetic rats, despite decreased glucose uptake and l-glycerol 3-phosphate concentration, flow of carbon atoms through l-glycerol 3-phosphate to glyceride glycerol was increased about threefold. 6. In fat pads, flow of carbon atoms through l-glycerol 3-phosphate to glyceride glycerol was increased by insulin (twofold), by adrenaline in the presence of insulin (fivefold) and by diabetes in pads incubated with insulin (1.5-fold). These increases could not be correlated either with increases in glucose uptake, which was unchanged by adrenaline and decreased in diabetes, or with the concentration of l-glycerol 3-phosphate, which was decreased by adrenaline and unchanged in diabetes. 7. These results are discussed in relation to the control of glyceride synthesis in heart and adipose tissue and to the regulation of glyceride fatty acid oxidation in the perfused rat heart.