The activities of lipases and carnitine palmitoyl-transferase in muscles from vertebrates and invertebrates

1. The activities of tri-, di- and mono-glyceride lipase and carnitine palmitoyltransferase were measured in homogenates of a variety of muscles. These activities were used to estimate the rate of utilization of glycerides and fatty acids by muscle. In muscles whose estimated rates of fat utilization can be compared with rates calculated for the intact muscle from such information as O₂ uptake, there is reasonable agreement between the estimated and calculated rates. 2. In all muscles investigated the maximum rates of hydrolysis of glycerides increase in the order triglyceride, diglyceride, monoglyceride. The activity of diglyceride lipase is highest in the flight muscles of insects such as the locust, waterbug and some moths and is lowest in the flight muscles of flies, bees and the wasp. These results are consistent with the utilization of diglyceride as a fuel for some insect flight muscles. 3. In many muscles from both vertebrates and invertebrates the activity of glycerol kinase is similar to that of lipase. It is concluded that in these muscles the metabolic role of glycerol kinase is the removal of glycerol produced during lipolysis. However, in some insect flight muscles the activity of glycerol kinase is much greater than that of lipase, which suggests a different role for glycerol kinase in these muscles.     

Lipid storage and changes during flight by triatomine bugs (Rhodnius prolixus and Triatoma infestans)

Exceptionally large amounts of lipid are stored in flight muscles of Rhodnius prolixus and Triatoma infestans (197 and 90 μmoles glyceride glycerol per g fresh weight respectively). The bulk of this lipid is in the form of triacylglycerol.A significant decrease in the muscle lipid occurs during the first hour of flight. Over the same period there is an increase in haemolymph lipid (particularly of diacylglycerol) which is taken to indicate the use of lipid from the fat body. The carbohydrate content of muscle and haemolymph is low, so it is likely that the supply of energy for flight is provided almost exclusively by the oxidation of fat. Oleate and palmitate are the major fatty acid components of lipid from both Triatoma and Rhodnius and are probably also the major fatty acids used for oxidation.Maturation of flight ability is temporally associated with the development of flight muscle size and increase in glyceride content.     

Carbohydrate and fatty acid titres during flight of the migrant noctuid moth,Anticarsia gemmatalis Hübner

Haemolymph concentrations of total carbohydrate and fatty acids were determined in velvetbean caterpillar (Anticarsia gemmatalis Hübner, Lepidoptera: Noctuidae) adult females throughout a 4-hr period of tethered flight. Total carbohydrate concentration decreased from approx. 30 to 10 μg/μl during the first 45 min of flight. Total fatty acid concentration increased from approx. 20 to 40 μg/μl during the first 60 min of flight and then declined to and stabilized at preflight levels. The decrease in wet weight (from approx. 97 to 80 mg/moth) during flight was probably due to defecation since no change in dry weight or haemolymph volume occurred. After 4 hr of flight, no apparent change in whole body lipid content (approx. 12 mg/moth) was observed but the much smaller carbohydrate content was reduced approx. 80% (from approx. 0.6 to 0.1 mg/moth). Approximately equal amounts (approx. 360–550 μg) of carbohydrate and lipid were removed from the haemolymph during 4 hr of flight. Changes in the haemolymph concentrations of palmitic, oleic and linoleic acids correspond to the changes in total fatty acid concentration of the haemolymph, indicating that these are the major components of the lipid mobilized and utilized during flight of A. gemmatalis.     

Metabolism of endogenous sterol ester by the superovulated rat ovary in vitro

Sterol, glyceride and phospholipid were found to account for more than 90% (w/w) of the lipid extracted from whole superovulated rat ovaries. These lipids, together with non-esterified fatty acids, were assayed in slices of the tissue after incubation for various times. Whereas the concentrations of triglyceride, diglyceride and phospholipid did not change significantly during incubation, that of sterol ester markedly decreased and those of free sterol, monoglyceride and non-esterified fatty acid increased. Evidence is presented that in this tissue (in contrast with other mammalian tissues) the main endogenous substrate for respiration is fatty acid derived from sterol ester.     

Piperine modulation of carcinogen induced oxidative stress in intestinal mucosa

The plasma-borne long-chain free fatty acids (FFA) enter skeletal muscle cells. Upon entering they are oxidized or esterified and a fraction remains free (non-esterified). The data on free fatty acids in skeletal muscles remain highly controversial. Furthermore, the composition of individual fatty acids in various lipid fractions including free fatty acids, monoglyceride and diglyceride in muscles has not been characterized. Also data on the composition of fatty acids esterified into muscle triglycerides and phospholipids are incomplete. The present study was undertaken to examine a composition of fatty acids in lipid fractions of different skeletal muscle types. For this purpose, samples of the rat soleus, red and white portions of gastrocnemius were excised, trimmed of visible fat and fascias and immediately frozen in liquid nitrogen. Samples were then pulverized and, lipids were extracted and fractionated by thin-layer chromatography. Individual long-chain fatty acids in different fractions were identified, characterized and quantitated by gas-liquid chromatography. FFA composition in the plasma was also determined. The total FFA content in the soleus, red and white gastrocnemius was 69.1 ± 10.8, 49.0 ± 13.6 and 22.7 ± 8.6 nmol/g, respectively. Palmitic and oleic acids were the major fatty acids in the muscles FFA fraction. Monoglyceride fraction of each muscle contained palmitic, stearic and linoleic acid as the major fatty acids, Diglyceride fraction contained mostly palmitic and oleic acid whereas triglyceride fraction mostly palmitic and linoleic acid.. The fraction of phospholipids was composed mostly of palmitic and linoleic acid but contained also considerable percentage of archidonic acid. Total plasma FFA/muscle FFA ratio depended on a muscle type and was: 2.4 in the soleus, 3.5 in the red and 7.4 in the white gastrocnemius. This assured transport of FFA to the myocytes. However, there were great differences in the ratio between particular FFA within the same muscle as well between the muscles. It indicates that individual FFA are either selectively transported from the plasma to the muscles or selectively used within the myocytes or both.     

Factors affecting concentrations of acetoacetate and d-3-hydroxybutyrate in haemolymph and tissues of the adult desert locust

The ketone bodies acetoacetate and d-3-hydroxybutyrate are found in the haemolymph, the fat body, and the flight muscles of the adult desert locust. Acetoacetate is the major ketone body in the haemolymph and the flight muscles, but in the fat body d-3-hydroxybutyrate usually predominates. The concentration of acetoacetate in the haemolymph varies with age, and increases during starvation and flight and also after the injection of corpus cardiacum homogenate; it is little affected by stress and there are no differences between the sexes. Ketone bodies appear to be formed in the fat body and are oxidized by the fat body, the flight muscles, and the testes. All the tissues oxidize acetoacetate much more readily than d-3-hydroxybutyrate, and the flight muscles of fed locusts oxidize acetoacetate much more readily than the fat body or the testes. In starved locusts the ability of the fat body and the flight muscles to oxidize ketone bodies is greatly reduced, but utilization by the testes remains normal. Thus the flight muscles appear to be the major consumers of ketone bodies in fed locusts, and the testes the major consumers in starved locusts. It is suggested that ketone bodies are formed in the fat body during the mobilization of the triglyceride lipid reserves, and are either oxidized by the fat body or transported by the haemolymph to the flight muscles and other tissues to be used as a respiratory fuel.     

Absorption and transport of dietary lipid in Pieris brassicae

The digestion and absorption of dietary glycerol tri(1-¹⁴C)oleate and oleic acid-1-¹⁴C and subsequent transport of the label was followed during the fifth instar in Pieris brassicae. The rate of incorporation of the label in tissue lipid was similar in both diets. Triolein was hydrolysed to free fatty acids (FFA), diglycerides (DGL), and monoglycerides (MGL). DGL were rapidly absorbed. FFA were less readily absorbed and some were excreted. In the gut wall the label was found in phospholipids (PL), triglycerides (TGL), DGL, and FFA. In haemolymph most of the label was in DGL and PL, but later appeared also in TGL and sterol esters (SE). The results suggest that DGL are released from the gut wall and carried in haemolymph into the fat body. In the fat body lipid is stored mainly as TGL, and released as DGL, TGL, and SE. The turnover of oleate in haemolymph DGL is rapid in comparison to haemolymph SE or TGL. Synthesis of PL in gut lumen is apparent. Much of this PL is excreted but some may be absorbed.     

Synthetic and modified glycerides: effects on plasma lipids

It has been suggested that the molecular species or structure of the triglyceride, i.e. not only what fatty acids are present but also their relative order in the sn1, 2, or 3 position on the triglyceride, can influence the metabolism of the triglyceride and its fatty acids, including lipoprotein metabolism. One rationale for this possibility assumes that the fatty acid in the sn2 position can be absorbed intact, i.e. as the sn2 monoglyceride, whereas the sn1,3 fatty acids are absorbed as free fatty acids that metabolize independently. Some sn2 monoglyceride might ultimately serve as the backbone for gut or liver phospholipids, exerting downstream influence on lipid metabolism. Experiments that test this hypothesis directly by feeding triglycerides with modified structure during carefully controlled fat intake are few, particularly in humans, but their results tend to support the paradigm.     

Dynamics of energy substrates in the haemolymph of Locusta migratoria during flight

In the two-fuel system for flight of the migratory locust, the haemolymph carbohydrate concentration falls during flight periods of up to 1 hr, the decrease being greater in case the pre-flight carbohydrate level is higher. The increase in the lipid concentration from the onset of flight is virtually independent of the initial lipid concentration. Flight intensity affects these changes in substrate concentrations: the carbohydrate level decreases more rapidly if flight speed is higher, whereas the increase in lipid concentration is delayed at higher flight speeds. Respiratory carbon dioxide production is elevated rapidly during flight and reaches over eight times the resting level. From the rate of ¹⁴CO₂ production after labelling of the haemolymph diglyceride pool it is concluded that diglycerides contribute to providing the energy for flight from the earliest stage of flying activity; diglyceride oxidation increases until maximum utilization is attained after some 45 min of flight. The decline in haemolymph carbohydrate concentration due to flying activity results in a decrease of haemolymph osmolarity. Free amino acids, particularly taurine, increase markedly in the haemolymph during flight; yet their concentration only partially counterbalances the fall in haemolymph osmolarity.     

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.     

Concentrations of glycerides and phospholipids in rat heart and gastrocnemius muscles: Effects of alloxan-diabetes and perfusion

1. Methods are described for the extraction of lipid and assay of mono-, di- and tri-glyceride glycerol and phospholipid phosphorus in rat heart and gastrocnemius muscles. 2. In hearts from normal animals, concentrations found were: monoglyceride, 0·6; diglyceride, 0·1; triglyceride, 12·6μmoles of glyceride glycerol/g. of dry muscle; phospholipid, 171μg.atoms of phospholipid phosphorus/g. of dry muscle. Concentrations of glycerides in gastrocnemius muscle were similar to heart muscle but those of phospholipids were lower (64μg.atoms of phospholipid phosphorus/g. of dry muscle). 3. Alloxan-diabetes increased the concentration of triglyceride in the muscles twofold. This increase was shown to be dependent in the heart on the availability of growth hormone and cortisol but not on the availability of dietary lipid. Total glyceride in the heart was increased after 48 and 72hr. starvation but not after 96hr. Changes in glyceride concentration seen in starvation and diabetes were not associated with significant changes in phospholipid concentration. It is suggested that mobilization of free fatty acids in diabetes leads to the synthesis of additional glyceride in muscle. 4. The possible contribution of glyceride fatty acid in the heart to respiration during perfusion has been calculated from the net loss of glyceride during perfusion, and also from the relative rates of lipolysis and esterification and compared with oxidation of fatty acid required for the balance of oxygen consumption (oxygen not utilized in the oxidation of glucose or glycogen glucose). In the normal or diabetic heart perfused with glucose and insulin the breakdown of glyceride can account for the balance of oxygen consumption. In the normal heart perfused without substrate the balance of oxygen consumption is not entirely accounted for by the breakdown of glyceride.     

Comparative analysis of the lipids of Acinetobacter species grown on hexadecane.

A comparative analysis of the cellular and extracellular lipids of Acinetobacter species HO1-N indicated basic physiological differences in hexadecane-grown cells. The cellular lipids obtained from hexadecane-grown cells were characterized by 3- and 18-fold increases in the phospholipid fraction and the mono- and diglyceride fraction, respectively, over that obtained from nutrient broth-yeast extract-grown cells. The cellular-associated pools of hexadecane were shown to comprise approximately 8% of the dry cell weight of hexadecane-grown cells. The extracellular lipids obtained from the culture broths of hexadecane-grown cells were comprised of triglyceride, mono- and diglyceride, free fatty acid, and wax ester. These lipids were either absent or present in minor concentrations in the culture broths of nutrient broth-yeast extract-grown cells. The exponential growth of Acinetobacter sp. on hexadecane was characterized by the significant accumulation of free fatty acid, monoglyceride, and diglyceride in the culture medium. Wax ester was shown to represent a minor portion of the extracellular lipids during the exponential growth phase, appearing in significant proportion only after the culture had entered the stationary phase of growth.     

Regulation of triglyceride synthesis in the parturient guinea-pig mammary gland

1. The specific activity of the enzyme palmitoyl-CoA-l-glycerol 3-phosphate palmitoyltransferase (EC 2.3.1.15) in the mammary tissue of guinea pigs has been shown to increase 37-fold at parturition. 2. Increases also occur in tissue concentrations of glycerol 3-phosphate, CoA and free fatty acid, but not in that of acid-insoluble CoA. 3. The isolation and fatty acid composition of plasma triglyceride and of mammary-tissue free fatty acid, diglyceride and triglyceride are described. 4. The findings are discussed in relation to the regulation of milk fat synthesis.     

Metabolic changes in the African fruit beetle,Pachnoda sinuata,during starvation

Specimens of the fruit beetle Pachnoda sinuata were starved for up to 30 days. The weight of the beetles declined consistently throughout the starvation period. Concentrations of carbohydrates and alanine in flight muscles, fat body and haemolymph decreased rapidly after onset of starvation, while the concentration of proline remained high. Whereas the lipid concentrations in the haemolymph did not change significantly upon starvation, the lipid content in flight muscles and fat body decreased significantly.Beetles that had been starved for 14 days responded to injection of Mem-CC, the endogenous neuropeptide from its corpora cardiaca, with hyperprolinaemia and a decrease in the alanine level, but no such effect was monitored after prolonged starvation of 28 days. Regardless of the period of starvation, Mem-CC injection could not cause hypertrehalosaemia or hyperlipaemia, although carbohydrates were increased in fed beetles after injection.Flight ability of beetles that had been starved for 15 or 30 days was apparently not impaired. During such periods, beetles used proline exclusively as fuel for flight as evidenced by the increase in the level of alanine in the haemolymph and decrease of the level of proline; the concentrations of carbohydrates and lipids remained unchanged.Activities of malic enzyme and alanine aminotransferase (enzymes involved in transamination in proline metabolism), glyceraldehyde-3-phosphate dehydrogenase (enzyme of glycolysis), 3-hydroxyacyl-CoA dehydrogenase (enzyme of beta-oxidation of fatty acids) and of malate dehydrogenase (enzyme of Krebs cycle) were measured in fat body and flight muscles. In flight muscle tissue the maximum activity of NAD(+)-dependent malic enzyme increased, while that of glyceraldehyde-3-phosphate dehydrogenase decreased during starvation, and malate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase and alanine aminotransferase were unchanged. In fat body tissue, activities of NADP(+)-dependent malic enzyme and 3-hydroxyacyl-CoA dehydrogenase increased during food deprivation and activities of glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase and alanine aminotransferase remained unchanged.     

Molecular characterization and association analysis of porcine adipose triglyceride lipase (<Emphasis Type="Italic">PNPLA2</Emphasis>) gene

The adipose triglyceride lipase (PNPLA2, also known as ATGL) is a novel triacylglycerol (TG) lipase which specifically removes the first fatty acid from the triglyceride molecule generating free fatty acid and diglyceride (DG) in mammalian cells. Here we describe the molecular characterization of the porcine ATGL gene. The full-length cDNA sequence contains a 1,461 bp open reading frame encoding a protein of 486 amino acids with a calculated molecular mass of 53.2 kDa and an isoelectric point of 7.90. The porcine ATGL protein shares high identity with other mammalian ATGL. The ATGL gene contains 9 coding exons, spans approximately 6 kb. The porcine ATGL mRNA was expressed predominantly in backfat, mildly in muscle, small intestine and heart, and almost absent in liver, spleen, lung, stomach, kidney and ovary. Statistical analysis showed the ATGL gene polymorphism (G/A³⁹²) was different between Chinese indigenous and introduced commercial western pig breeds, and was highly associated with almost all the fat deposition and carcass traits, including subcutaneous fat thickness, viscera adipose tissue, lean percentage, loin eye traits and even rib numbers.     

The metabolism of glycerol in the locust Schistocerca gregaria during flight

The concentration of glycerol in locust haemolymph increases 10-fold during 1 hr flight but decreases rapidly when flight ceases. [¹⁴C]Glycerol is rapidly metabolized by locusts in vivo. Trehalose and diacyl glycerol are the main products to appear in the haemolymph but the proportion of diacyl glycerol is increased in flown insects or when adipokinetic hormone is injected. Trehalose and diacyl glycerol are also the main products formed when isolated fat body is incubated with [¹⁴C]glycerol. Adipokinetic hormone increases the proportion of diacyl glycerol formed.It is proposed that during flight glycerol is produced by hydrolysis of diacyl glycerol in the flight muscles. It is then transported to fat body for esterification with fatty acid produced during conversion of triacyl glycerol stores to diacyl glycerol.     

Effect of Ca on composition of fat body of peanut seed

Peanut fruits were grown in nutrient media with or without Ca and in a soil with two Ca levels, from the 20th day after penetration of the gynophore. Seed weight was smaller in the nutrient medium without Ca than in the nutrient medium with Ca, and it was also smaller in the soil with 4 meq of exchangeable Ca (L treatment) than in the soil with 10 meq of exchangeable Ca (H treatment). The fat body of seeds from the Ca deficient medium and the L treatment had a decreased phospholipid content and an increased simple lipid content. In the seed from the H treatment, phosphatidylcholine increased from the 30th to 60th day, while caldiolipin decreased. The amount of triglyceride in the simple lipid content of fat body was decreased by Ca deficiency in the nutrient medium, whereas that of diglyceride was increased, but these effects were not observed in the fat body of the seed from the L treatment. No effect of Ca deficiency was observed in the fatty acid composition of triglyceride.     

The molecular and metabolic essentials for long-distance flight in insects

Summary The mechanism of long-distance flight in insects was investigated by comparing lipid mobilization and transport in gregarious- and solitary-phase locusts and in the American cockroach. Unlike the gregarious-phase locust, both the American cockroach and the solitary locust were unable to form low-density lipophorin (loaded with increased amount of diacylglycerol) even when injected with adipokinetic hormone (AKH). The cockroach fat body responded to AKH. However, not only does the American cockroach lack apolipophorin-III (apoLp-III) in the haemolymph, but the fat body contains only an extremely small amount of diacylglycerol and a relatively large triacylglycerol pool. By contrast, the solitary-phase locust had apoLp-III in the haemolymph, but the fat body was only one-seventh or less in weight of the fat body of the gregarious locust. Furthermore, the fat body of the solitary locust contains a very small amount of triacylglycerol (1/20 or less of that of the gregarious locust) with only a trace of diacylglycerol. It was concluded that in the American cockroach and the solitary locust, the stores of fuel in the fat body are insufficient to maintain prolonged flight.Abbreviations AKII adipokinetic hormone - apoLp-III apolipophorin III - HDLp high-density lipophorin - LDLp low-density lipophorin - LTP lipid transfer particle - MW molecular weight - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Specificity and localisation of lipoprotein lipase in the flight muscles of Locusta migratoria

Using natural lipoproteins as substrates, lipase activity has been measured in leg muscle, fat body, midgut and flight muscles of Locusta migratoria. The enzymic activity in the flight muscles is higher than in those other tissues tested, confirming the potential of the flight muscles to utilise lipids at high rates. In addition, a membrane-bound lipoprotein lipase can be extracted from flight muscle. The flight muscle enzyme activity shows a marked substrate specificity; at lipoprotein concentrations equivalent to those found normally in flown or resting locusts respectively, the enzyme hydrolyses diacylglycerols associated with lipoprotein A+ (present in the haemolymph of flown or adipokinetic hormone-injected locusts) at about 4 times the rate of those associated with lipoprotein Ayellow (which is the major lipoprotein in resting locusts). In addition, the hydrolysis of lipids carried by lipoprotein Ayellow is dramatically reduced in the presence of lipoprotein A+. These observations indicate that the enzyme plays a specific role in the uptake of lipids at the flight muscles to ensure a smooth transition from carbohydrate to lipid based metabolism during flight.     

Utilization of endogenous lipid by the isolated perfused rat heart

1. The lipids of the rat heart have been studied with regard to amount, classes present and fatty acid composition of free fatty acids, triglycerides and phospholipids. Myocardial lipid contained 300μmoles of total fatty acid/g. dry wt. of which only 2–4μmoles were free; the remainder was esterified, chiefly as phospholipid. Neutral esters, of which triglyceride was the principal form, made up 15% of the total fatty acids. 2. When normal hearts were perfused with a nutrient-free medium until exhaustion, the triglyceride concentration declined from 43 to 13μmoles/g. dry wt. The content of phospholipids, partial glycerides and cholesteryl esters did not change. When the lipids of the rat heart were labelled with [1-¹⁴C]palmitate before perfusion with non-nutrient medium, radioactivity disappeared from the triglyceride, diglyceride and free fatty acid fractions, but not from the phospholipid or other ester classes. 3. These experiments support the view that only a small fraction of the total cardiac lipid, principally triglycerides and to a smaller extent diglycerides, is available as a source of fuel in the absence of exogenous substrate.