Fat Cake Research Articles

Diacylglycerol transport in the insect fat body: evidence of involvement of lipid droplets and the cytosolic fraction

In this report we show the existence of a distinct pool of fat body diacylglycerol (DG) that can be distinguished from the bulk DG. This is a dynamic pool of DG that uses FA entering the fat body from the hemolymph, whereas the bulk DG uses the fatty acids stored in the fat body fat droplets. Using a dual labeling technique, it was possible to compare the effect of hormone-stimulated DG synthesis and secretion on the distribution of radiolabeled FA among the lipids of the dynamic pool (short-term radiolabeling), with the hormonal effect on the total complement of fat body lipids (long-term radiolabeling). We observed that, whereas DG represents 2% to 3% of the fat body lipid mass, about 20% of the short-term radiolabeled lipids are represented by DG. Stimulation of lipolysis produces a fast decrease in the fraction of short-term radiolabeled DG, whereas there is an increase in the mass of fat body DG. The subcellular distribution of bulk DG showed that its majority (62%) was in the fat cake whereas only 2.9% was in the cytosol. On lipolysis stimulation, the largest changes in specific activities of newly synthesized DG were detected in the cytosol and the fat cake, suggesting that newly synthesized DG localized in the lipid droplets and the cytosol is preferentially mobilized. —Arrese, E. L., J. L. Gazard, M. T. Flowers, J. L. Soulages, and M. A. Wells. Diacylglycerol transport in the insect fat body: evidence of involvement of lipid droplets and the cytosolic fraction.

Characterization of a platelet activating factor acetylhydrolase from rat adipocyte

Platelet activating factor-acetylhydrolases (PAF-AHs) are a family of enzymes with the common property of hydrolyzing and inactivating PAF and thus regulating its levels. In the course of studying the role of PAF in rat adipocytes and its possible implication in body weight regulation and immune response, conditions in which adipocytes are involved, we investigated the existence of PAF-AH in these cells. We detected PAF-AH activity in rat adipocytes which is mainly distributed in the cytosol. The behaviour of the enzyme during hydrophobic chromatography, together with the fact that part of the enzyme activity was found in the fat cake of adipocyte homogenate suggests the hydrophobic nature of rat adipocyte PAF-AH. The enzyme activity was distinct from the Ca 2+-dependent and independent phospholipase A 2, the lysophospholipase, the lecithin:cholesterol acyltransferase (LCAT) and from non-specific acetylhydrolases. We partially purified PAF-AH from rat adipocyte cytosolic fraction and the purified enzyme revealed a major protein band of 66 kDa and a minor one of 37 kDa on SDS/PAGE. The purified PAF-AH has an apparent K m value of 4.9 μM and the enzyme activity was inactivated by PMSF and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and moderately stimulated by dithiothreitol (DTT). Furthermore, in this study we identified PAF in rat adipocytes and determined its concentration.

Mechanism of anti-lipolytic action of acipimox in isolated rat adipocytes.

Acipimox is commonly used to treat hypertriglyceridaemia in non-insulin-dependent diabetic patients, but its precise mechanism of action has yet to be elucidated. We examined the in vitro effects of acipimox on the lipolytic regulatory cascade in epididymal adipocytes isolated from Wistar rats. Acipimox inhibited the lipolytic rate stimulated by adenosine deaminase (1 U/ml) in a concentration-dependent manner, reaching a near-basal value at 10 mumol/l acipimox. Lipolysis activated by sub-maximal levels of isoproterenol in combination with adenosine deaminase (20 mU/ml) was significantly (p < 0.05) decreased by 100 mumol/l acipimox, whereas, in the absence of adenosine deaminase, 100 mumol/l acipimox showed no significant (p > 0.05) inhibition. These findings suggested that the anti-lipolytic mechanism regulated by adenosine may also be regulated by acipimox. Acipimox diminished the intracellular cyclic AMP level produced by 25 nmol/l isoproterenol in the presence of adenosine deaminase (20 mU/ml) in a concentration-dependent manner. At the same level of stimulation, acipimox inhibited the cyclic AMP-dependent protein kinase activity ratio and lipolytic rate over the same concentration range, with significant (p < 0.05) reductions occurring at and above, 0.5 mumol/l and 10 mumol/l acipimox, respectively. Western blotting showed that upon lipolytic stimulation (1 U/ml adenosine deaminase; 100 nmol/l isoproterenol) a threefold increase in the lipolytic rate was accompanied by a significant (p < 0.05) rise in hormone-sensitive lipase associated with the lipid fraction. Acipimox (1 mmol/l) and insulin (1 nmol/l) re-distributed hormone-sensitive lipase back to the cytosol, with a corresponding significant (p < 0.05) loss from the fat cake fraction of adipocyte homogenates. In conclusion, the anti-lipolytic action of acipimox is mediated through suppression of intracellular cyclic AMP levels, with the subsequent decrease in cyclic AMP-dependent protein kinase activity, leading to the reduced association of hormone-sensitive lipase with triacylglycerol substrate in the lipid droplet of adipocytes.

Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet.

Hormone-sensitive lipase activity (HSL), which is found in the supernatant of centrifuged homogenates of lipolytically quiet isolated rat adipocytes, was greatly reduced in or absent from the supernatant of lipolytically stimulated cells. The lipase was purified 100- to 250-fold from the supernatant of lipolytically quiet cells to 10-20% purity by a single passage over phenyl-Sepharose resin with high (greater than 70%) activity yields. Western blotting of adipocyte homogenate fractions with polyclonal antiserum raised against HSL showed that the enzyme shifted quantitatively from the supernatant of control cells to the floating "fat cake" of lipolytically stimulated cells. A similar shift to the fat cake was observed when cells were disrupted by hypotonic lysis and centrifugation rather than by homogenization. We propose that upon lipolytic activation of adipocytes and phosphorylation of HSL by cAMP-dependent protein kinase, the critical event is not an increase in catalytic activity (i.e., turnover number) but a translocation of the lipase to its substrate at the surface of the lipid storage droplet.

Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets

The lipid fraction ("fat cake") of rat epididymal adipocytes contains a prominent phosphoprotein (62 kDaapp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) that is multiply phosphorylated by cAMP-dependent protein kinase in vivo, at which point it migrates as a 65/67-kDaapp doublet by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is by far the most heavily radiolabeled protein in the cell. Western blot analysis of various tissues with immunopurified antibodies purified from antisera raised against the 62-kDa species suggests that the protein is specific for adipocytes. This protein, which we term perilipin, is found in differentiated cultured 3T3-L1 adipocytes, but not in their precursor 3T3-L1 fibroblasts. Immunocytochemical studies with specific antiserum shows that the perilipin is closely associated with the periphery of lipid storage droplets in cultured adipocytes. Given its adipocyte specificity, acute regulation by hormones, and subcellular location, we speculate that perilipin plays a role in the specialized lipid storage function of adipocytes.

Dry extrusion as an aid to mechanical expelling of oil from soybeans

AbstractA new concept is described for mechanical extraction of oil from soybeans, using dry extrusion as a pretreatment. It was found that coarsely ground whole soybeans at 10 to 14% moisture could be extrusion cooked so that the extrudate emerges from the die in a semi‐fluid state. The dwell time within the extruder was less than 30 seconds, and the temperature was raised to about 135 C. The semi‐fluid extrudate was immediately pressed in a continuous screw press to obtain high quality oil and press cake. Extrusion prior to expelling greatly increased the throughput of the expeller over the rated capacity. An oil recovery of 70% was obtained in single pass expelling using pilot model expellers. Higher recovery rates can be expected with commercial scale expellers. The high temperature‐short time extrusion cooking process eliminates the prolonged heating and holding of raw material in conventional expelling. Under the experimental conditions, press cake with 50% protein, 6% residual oil and 90% inactivation of trypsin inhibitors was obtained. The low fat cake was easily ground in a hammer mill without the usual problems associated with milling of whole beans. The expelled oil was remarkably stable with an AOM stability of 15 hr, which is comparable to refined deodorized oil according to NSPA specifications. The new procedure offers potential for producing natural soybean oil and food grade low fat soy flour by a relatively low cost operation. It may be adopted as an improvement to existing conventional expelling operations in less developed countries or as a commercial or on‐farm operation for producing value added products from soybeans within the U.S.

Fat associated with adipose lipase the newly synthesized fraction that is the preferred substrate for lipolysis

The incorporation of fatty acids by adipocyte fat cake was observed. The newly formed lipid was not extracted by petroleum ether but remained in the lipase-containing residue. It was the first lipid to be hydrolyzed when the lipase was activated by exposure to the aqueous phase. The data presented indicate that the lipid fraction of the fat layer residue is the active compartment of cell acylglycerol and demonstrate that the compartmentation of acylglycerols into metabolic pools involves a physical separation between them.

Lipolytic activity in adipocyte cell fractions

Adipocytes release only negligible amounts of free fatty acids unless stimulated, but reveal considerable lipolytic activity when homogenized. Epinephrine treatment of the cells caused only a 20–40% increase in the activity of infranatants of homogenates while raising the activity associated with the fat layer up to 10-fold. Full activity (i.e. that of intact-activated cells) could be revealed by epinephrine treatment of the homogenate as well as by sonication of the fat layer in buffer. The combination of both treatments did not yield higher activities. The fat cake contains the bulk of the potential activities which are only realized when dispersed in the aqueous phase by sonication, or upon hormone activation of the whole homogenate. Increase in activity could also be obtained by removal of most of the lipid from the fat layer by extraction with petroleum ether. Re-introduction of extracted lipid inhibited lipolysis. The active enzyme could be separated by flotation at 1.12 specific gravity. The data suggest that the lack of activity in the intact non-stimulated cell may be due to the lack of availability of the aqueous phase to the enzyme.

Action of insulin and catecholamines on the phosphorylation of proteins associated with the cytosol, membranes, and “fat cake” of rat fat cells.

Action of insulin and catecholamines on the phosphorylation of proteins associated with the cytosol, membranes, and “fat cake” of rat fat cells.

Subcellular Distribution of and Epinephrine-induced Changes in Hormone-sensitive Lipase, Phosphorylase, and Phosphorylase Kinase in Rat Adipocytes

Abstract A systematic study was carried out of the subcellular distribution of hormone-sensitive lipase, glycogen phosphorylase, and phosphorylase kinase in rat adipocytes and the changes in these enzymes when the cells were incubated with epinephrine. Under the conditions of homogenization used, approximately one-third of the total hormone-sensitive lipase activity was associated with the fat cake floated to the top of a homogenate by low speed centrifugation, although as discussed in detail, this value remains uncertain in view of technical problems faced in assaying lipase tightly bound to fat. Subfractionation of the fat-free homogenate showed that over 80% of the hormone-sensitive lipase was recovered in the soluble fraction and the specific activity of that fraction was considerably higher than it was in particulate fractions. The plasma membrane fraction contained less than 2% and the microsomal fraction less than 3% of the activity in the fat-free homogenate. When adipocytes were lysed, over 80% of the total lipase activity was recovered in the supernatant fraction and less than 20% in the fat cell ghosts. The distribution of lipase activity among subcellular fractions of the fat-free homogenate was not altered by previous treatment of the adipocytes with epinephrine. Lipase activity in the soluble fraction from control cells was activated by treatment with cyclic adenosine 3',5'-monophosphate-dependent protein kinase and MgATP. When the cells had been previously treated with epinephrine, the percentage of activation by protein kinase was reduced in this and in all fractions. Phosphorylase and phosphorylase kinase activity were found predominantly in the soluble fraction; small amounts (21 and 14%, respectively) were associated with the fat cake assayed after resuspension in Triton X-100. The distribution of these enzymes in cells previously treated with epinephrine was not significantly different from that in control cells. Total phosphorylase activity (assayed in the presence of 5'-AMP) was significantly increased in homogenates prepared from cells previously treated with epinephrine as was the ratio of phosphorylase activities measured in the absence and in the presence of 5'-AMP. On the other hand, there was no change in total phosphorylase kinase activity after epinephrine treatment and no change in the ratio of activities measured at low pH values (6.2 or 6.8) and high pH value (8.2). Further characterization of the phosphorylase and phosphorylase kinase systems in adipose tissue is needed to assess the regulatory role of phosphorylase kinase in this tissue.

Studies on the hormone-sensitive lipase of adipose tissue

Sucrose gradient centrifugation has been used to examine the triglyceride lipases present in extracts of rat epididymal adipose tissue. The aqueous infranatant recovered between the pellet and fat cake of tissue homogenates which had been centrifuged at 40,000 g was shown to contain two types of triglyceride lipase activity. One of these appears in the 15s region and has been identified as the active form of the "hormone-sensitive lipase" believed to be responsible for initiating the hydrolysis of tissue triglyceride stores in response to lipolytic stimuli. The activity of this enzyme was selectively increased in extracts prepared from tissue exposed to epinephrine and decreased in extracts of insulin-treated tissue. The increased lipolytic activity of extracts of tissue from fasted or fasted-refed rats was also found largely in this region. When the tissue was incubated with orthophosphate-(32)P, radioactivity was incorporated into a protein migrating at 15s. A second peak of triglyceride lipase activity appeared in the 6s region coincident with the location of the monoglyceride and diglyceride lipase activities. The amount of 6s triglyceride lipase activity did not correlate with changes in the lipolytic activity of the tissue from which the extracts were prepared, and its physiological function remains to be elucidated. The lipoprotein lipase and the short-chain triglyceride lipase ("tributyrinase") each moved more slowly in the gradient than the 6s triglyceride lipase. Both the 6s and 15s enzymes were shown to be present in washed adipocytes isolated from the tissue by collagenase digestion.

Properties of a hormone-stimulated lipase from rat adipose cells

A well defined, easily reproducible system for the study of hormone-stimulated lipolysis in broken cell preparations from rat adipocytes has been described. Addition of hormone prior to cellular rupture results in a homogenate which liberates free fatty acids (FFA) in a linear fashion for at least 1 hr. The quantity of FFA liberated from nonhormone-treated control homogenates is much smaller but is also linear for at least 1 hr. The activation process occurring after addition of hormone appears to be complete within 1 min. Preincubation of the cells prior to addition of the hormone does not increase the magnitude of the response to the hormone. Broken cell preparations made from both control and hormone-treated cells show a pH optimum in the range of 6.5-1.5, and both preparations show a necessity for the presence of bovine serum albumin in the assay medium. The centrifugation of an activated homogenate at 0° and subsequent recombination of the resulting fractions (fat cake, infranatant and pellet) produced a preparation showing lipolysis. When the infranatant from an untreated homogenate was recombined with the fat cake from a hormone-treated homogenate, hormone-stimulated lipolysis was still seen; the reverse combination showed no such stimulation of lipolytic activity. When the infranatant fluid was removed entirely and replaced with warm (37°) Krebs-Ringer phosphate buffer (KRP), lipolysis was greater in hormone-treated than in control preparations, and the specific activity was increased 5-fold. It is tentatively concluded, therefore, that the “active” lipolytic properties are associated with the fat cake; the infranatant fluid appears to inhibit the lipolytic process.

Lipase activity in the fat cake and aqueous phase of adipose tissue homogenate of fed, fasted and fasted-refed rats.

Abstract. 1. Free fatty acid production by the fat cake of adipose tissue was found to be much greater in fasted‐refed than in fasted and fed rats, respectively. Preincubation with adrenalin and dibutyryl cyclic AMP stimulated lipase activities in fasted and fed rats, but not in fasted‐refed rats. Basal lipase activity in the fat cake of fasted‐refed rats was higher than adrenalin or dibutyryl cyclic AMP activated lipase activity of fasted or fed rats. Activation of lipase activity by refeeding lasted for several days.—2. Lipase activity in the aqueous phase of adipose tissue homogenate of all rats was in the same range.—3. Free fatty acid release during the assay of lipase in the fat cake proceeded for a period of up to 120 minutes although not in an entirely linear manner. The ratio of free fatty acids to glycerol release was 2.9. The pH optimum of the reaction was 7.4. NaCl, NaF, protamine and heparin markedly inhibited the lipase reaction. Heparin in the preincubation medium did not reduce the lipase activity in the fat cake.— 4. The very active triglyceride lipase activity of adipose tissue of fasted‐refed rats explains earlier findings showing that intact adipose tissue of fasted‐refed rats exhibits a very high glycerol release. It appears that tissue levels of triglyceride lipase increase during refeeding under the influence of insulin, i.e. at a time when the level of cyclic AMP in the tissue supposedly is low.