STRUCTURE-FUNCTION RELATIONSHIPS IN THE ADIPOSE CELL : III. Effects of Bovine Serum Albumin on the Metabolism of Glucose and the Release of Nonesterified Fatty Acids and Glycerol by the Isolated Adipose Cell

Serum albumin stimulates the uptake of U-glucose-¹⁴C and the incorporation of ¹⁴C-counts into triglyceride glycerol and inhibits the incorporation of ¹⁴C-counts into triglyceride fatty acids by isolated adipose cells; insulin and epinephrine enhance these effects. In the absence of hormones, these responses to albumin increase with increasing albumin concentration. In the presence of insulin, a qualitatively similar pattern of increasing responses to albumin is observed; the enhancement of each response by insulin is, however, only slightly potentiated by higher albumin concentrations. In contrast, in the presence of epinephrine, these responses to albumin are maximal at the lowest albumin concentration tested, 0.1%; the enhancement of each response by epinephrine is similarly maximal at 0.1% albumin, but decreases rapidly as the albumin concentration is raised. Increasing serum albumin concentrations do, however, stimulate the release of fatty acids and glycerol by epinephrine-treated cells increasingly until a plateau, determined by the epinephrine dose, is reached. These data support the suggestion that intracellular fatty acid levels function in the regulation of adipose cell activity, and further suggest that serum albumin plays a role in determining the metabolic fate of these fatty acids.     

STRUCTURE-FUNCTION RELATIONSHIPS IN THE ADIPOSE CELL : I. Ultrastructure of the Isolated Adipose Cell

A method is described for preparing isolated rat adipose cells for electron microscopy. The ultrastructure of such cells and their production of ¹⁴CO₂ from U-glucose-¹⁴C were studied simultaneously in the presence of insulin or epinephrine. Each adipose cell consists of a large lipid droplet surrounded by a thin rim of cytoplasm. In addition to typical subcellular organelles, a variety of small lipid droplets and an extensive system of membranes characterize the cell's cytoplasm. A fenestrated envelope surrounds the large, central lipid droplet. Similar envelopes surround cytoplasmic lipid droplets occurring individually or as aggregates of very small, amorphous droplets. Groups of individual droplets of smaller size also occur without envelopes. The system of membranes consists of invaginations of the cell membrane, vesicles possibly of pinocytic origin, simple and vesiculated vacuoles, vesicles deeper in the cytoplasm, flattened and vesicular smooth surfaced endoplasmic reticulum, and Golgi complexes. Neither insulin nor epinephrine produced detectable ultrastructural alterations even when cells were incubated under optimal conditions for the stimulation of ¹⁴CO₂ evolution. Structural responses of the isolated adipose cell to hormones, if such occur, must, therefore, be dynamic rather than qualitative in nature; the extensive system of smooth surfaced membranes is suggestive of compartmentalized transport and metabolism.     

A comparative study of the effects of polyamino acids and dextran derivatives on pinocytosis in the rat yolk sac and the rat peritoneal macrophage

Poly-l-lysine, poly-l-α-ornithine, poly-l-glutamic acid, dextran, DEAE-dextran and dextran sulphate all fail to affect greatly the rate of pinocytic uptake of ¹²⁵I-labelled polyvinylpyrrolidone by 17.5-day rat visceral yolk sac or rat peritoneal macrophages cultured in vitro. It is concluded that these agents do not much affect the rate of pinocytic ingestion of liquid. Polycations accelerate the accumulation of colloidal ¹⁹⁸Au in both systems, and this is ascribed to the formation of substrate · modifier complexes which either adsorb to plasma membrane, and thus gain rapid entry, or initiate another mode of endocytosis. Pinocytic uptake of formaldehyde-denatured ¹²⁵I-labelled bovine serum albumin was accelerated by poly-l-lysine and poly-l-ga-ornithine in the macrophage, buth inhibited in the yolk sac.     

Adipose tissue lipolysis is increased during a repeated bout of aerobic exercise.

The goal of the study was to examine whether lipid mobilization from adipose tissue undergoes changes during repeated bouts of prolonged aerobic exercise. Microdialysis of the subcutaneous adipose tissue was used for the assessment of lipolysis; glycerol concentration was measured in the dialysate leaving the adipose tissue. Seven male subjects performed two repeated bouts of 60-min exercise at 50% of their maximal aerobic power, separated by a 60-min recovery period. The exercise-induced increases in extracellular glycerol concentrations in adipose tissue and in plasma glycerol concentrations were significantly higher during the second exercise bout compared with the first (P < 0.05). The responses of plasma nonesterified fatty acids and plasma epinephrine were higher during the second exercise bout, whereas the response of norepinephrine was unchanged and that of growth hormone lower. Plasma insulin levels were lower during the second exercise bout. The results suggest that adipose tissue lipolysis during aerobic exercise of moderate intensity is enhanced when an exercise bout is preceded by exercise of the same intensity and duration performed 1 h before. This response pattern is associated with an increase in the exercise-induced rise of epinephrine and with lower plasma insulin values during the repeated exercise bout.     

Lipoprotn lipase content and triglyceride fatty acid uptake in adipose tissue of rats of differing body weights

Increasing body weight appears to alter lipid metabolism in adipose tissue. We have measured the content of lipoprotein lipase and the uptake of chylomicron triglyceride fatty acids in epididymal fat pads of rats of different weights. In order that the results might be expressed in terms of cell numbers, the relationship between the weights of fat pads and the numbers and volumes of fat cells isolated from them was determined. Highly significant correlations were found between fat pad weight and both the number and the volume of the individual adipocytes. In rats weighing from 140 to 350 g, the increase in the size of fat pads was attributable almost equally to increases in cell size and in cell number. Lipoprotein lipase activity was measured in acetone powders of whole fat pads and of isolated fat cell preparations. With both, lipoprotein lipase activity per cell diminished significantly as the weight of fat tissue increased, i.e., larger fat cells contained less enzyme per cell than smaller cells. The uptake of triglyceride fatty acid radioactivity was measured after incubation of fat pads with radiolabeled rat lymph chylomicrons in flasks containing either buffer alone or with added glucose or glucose plus insulin. The addition of glucose and insulin led to a mean increase of 70% in the uptake of radioactivity, but larger adipocytes were stimulated less than smaller cells. This resulted in a significant negative correlation between the weights of fat pads and the uptake of radioactivity. Enlargement of fat cells also led to a diminution in their capacity to esterify fatty acids.     

Pinocytic uptake of divinyl ether-maleic anhydride (pyran copolymer) and its failure to stimulate pinocytosis

The effect of DIVEMA (pyran copolymer) and three DIVEMA derivatives on the pinocytic uptake of ¹²⁵I-labelled PVP and colloidal ¹⁹⁸Au by the rat visceral yolk sac and by rat peritoneal macrophages was studied in vitro. Contrary to expectations from some earlier data, there was no enhancement of pinocytosis and in some cases inhibition was seen. [¹⁴C]DIVEMA and ¹²⁵I-labelled DIVEMA were accumulated rapidly by rat peritoneal macrophages, the results indicating that this is by an adsorptive pinocytic mechanism.     

Pinocytosis in the rat visceral yolk sac effects of temperature, metabolic inhibitors and some other modifiers

Low temperature, 2,4-dinitrophenol and moniodoacetate could each completely abolish the pinocytic uptake of ¹²⁵I-labelled polyvinylpyrrolidone, ¹²⁵I-labelled bovine serum albumin or colloidal ¹⁹⁸Au by 17.5-day rat visceral yolk sac cultured in vitro. Cytochalasin B and colchicine caused a partial and dose-dependent inhibition. It is concluded that the mechanism of pinocytic uptake of these substrates is not micropinocytosis as conventionally defined. Removal of extracellular calcium or the oresence of theophylline inhibited liquid-phase pinocytosis by the rat yolk sac, whereas addition of ouabain caused a biphasic response: a slight stimulation of pinosome formation at a low concentration, and an inhibitory effect at a higher concentration.     

Growth Retardation, Impaired Triacylglycerol Catabolism, Hepatic Steatosis, and Lethal Skin Barrier Defect in Mice Lacking Comparative Gene Identification-58 (CGI-58)

Comparative gene identification-58 (CGI-58), also designated as α/β-hydrolase domain containing-5 (ABHD-5), is a lipid droplet-associated protein that activates adipose triglyceride lipase (ATGL) and acylates lysophosphatidic acid. Activation of ATGL initiates the hydrolytic catabolism of cellular triacylglycerol (TG) stores to glycerol and nonesterified fatty acids. Mutations in both ATGL and CGI-58 cause “neutral lipid storage disease” characterized by massive accumulation of TG in various tissues. The analysis of CGI-58-deficient (Cgi-58^−/−) mice, presented in this study, reveals a dual function of CGI-58 in lipid metabolism. First, systemic TG accumulation and severe hepatic steatosis in newborn Cgi-58^−/− mice establish a limiting role for CGI-58 in ATGL-mediated TG hydrolysis and supply of nonesterified fatty acids as energy substrate. Second, a severe skin permeability barrier defect uncovers an essential ATGL-independent role of CGI-58 in skin lipid metabolism. The neonatal lethal skin barrier defect is linked to an impaired hydrolysis of epidermal TG. As a consequence, sequestration of fatty acids in TG prevents the synthesis of acylceramides, which are essential lipid precursors for the formation of a functional skin permeability barrier. This mechanism may also underlie the pathogenesis of ichthyosis in neutral lipid storage disease patients lacking functional CGI-58.     

The quantitative estimation of pinocytosis using radioactive colloidal gold

Radioactive colloidal gold has been used as a quantitative indicator of the pinocytic activity of mouse peritoneal macrophages. The uptake of label is both time and temperature dependent, accumulation being linearly related to time for several hours. Uptake is also dependent on the number of cells and concentration of the radioactive label. Increasing concentrations of newborn calf serum in the culture medium cause dramatic increases in pinocytosis without corresponding increases in protein levels in the cells.     

Pinocytic stimulation in Dictyostelium discoideum by γ-benzene hexachloride

Pinocytic activity is greatly stimulated in γ-BHC (gamma isomer of benzene hexachloride) treated, vegetative cells of Dictyostelium discoideum as measured by ¹⁴C sucrose or FITC-dextran uptake. Transmission electron microscopic studies also reveal the presence of a greater number of pinosomal vesicles in the pesticide-treated Dictyostelium amoebae. The enhanced pinocytic activity has been discussed in relation to lipophilic interactions of γ-BHC with the hydrophobic cell surface and the observed changes in the cytoskeletal proteins of the treated cells. © 1994 Wiley-Liss, Inc.     

Cell-associated nonesterified fatty acid levels and their alteration during lipolysis in the isolated mouse adipose cell

A rapid and flexible method has been developed for measuring cell-associated, probably intracellular, nonesterified fatty acids (CAFA) in isolated mouse adipose cells. A variety of lipolytic agents as well as various concentrations of epinephrine elevate CAFA levels in rough proportion to their stimulation of glycerol and fatty acid release. Insulin reduces epinephrine-elevated CAFA levels. A detailed, quantitative study of the relationship among lipolytic activity, CAFA levels, and the extracellular molar ratio of fatty acids to albumin has been carried out. Epinephrine-elevated CAFA levels rise linearly with, while epinephrine-stimulated lipolytic activity is independent of, fatty acid to albumin ratios below 2-3. As the ratio increases from 3 to 5, CAFA levels continue to increase, whereas lipolytic activity decreases. Above ratios of 5, fatty acid release almost completely ceases; CAFA levels increase dramatically with residual glycerol release. A temperature-dependent efflux of epinephrine-elevated CAFA can be elicited through blockade of stimulated lipolysis with propranolol, but only in the presence of extracellular fatty acid to albumin ratios below 3. These observations suggest that during stimulated lipolysis, a fatty acid gradient exists between the cell and extracellular serum albumin and that CAFA represent the intracellular component of this gradient. In addition, these observations support the concept that intracellular fatty acids play a role in the feedback regulation of adipose cell function as extracellular fatty acids accumulate during the lipolytic response.     

Integrative metabolic regulation of peripheral tissue fatty acid oxidation by the SRC kinase family member Fyn

Mice null for Fyn (a member of the Src family of nonreceptor tyrosine kinases) display a reduced percentage of adipose mass associated with decreased adipocyte cell size. In parallel, there is a substantial reduction in fasting plasma glucose, insulin, triglycerides, and free fatty acids concomitant with decreased intrahepatocellular and intramyocellular lipid accumulation. Importantly, the Fyn null mice exhibit improved glucose tolerance resulting from increased peripheral tissue (adipose and skeletal muscle) insulin sensitivity with a very small effect in the liver. Moreover, whole-body, adipose, and skeletal muscle fatty acid uptake and oxidation are increased along with AMP kinase activation and acetyl-CoA carboxylase inhibition. Together, these data demonstrate crosstalk between Src-family kinase activity and fatty acid oxidation and show that the loss of Fyn markedly improves peripheral tissue insulin sensitivity by relieving a selective negative modulation of AMP kinase activity in adipose tissue and skeletal muscle.     

Intermediary metabolism of adipose tissue.

Metabolism of ruminant adipocytes involves the synthesis and mobilization of lipids. Rates of lipid synthesis from the uptake of preformed fatty acids (via lipoprotein lipase) and de novo synthesis of fatty acids are related to the energy balance. Acetate is the major carbon source for fatty acid synthesis with NADPH originating from the pentose cycle and the isocitrate cycle. Ruminant adipose tissue lacks the ability to utilize for lipogenesis those substrates that generate mitochondrial acetyl CoA because of an absence of ATP citrate-lyase and NADP-malate dehydrogenase. Lipid mobilization in ruminant adipocytes is apparently regulated via cAMP levels and a summary of the compounds investigated for lipolytic responses is presented. The control of lipid synthesis and mobilization is interrelated in ruminant adipose tissue. The coordinated manner in which these two functions are regulated is examined with regard to adipocyte responses to insulin and epinephrine. In both lipid synthesis and lipid mobilization, ruminant adipocytes are uniquely different from nonruminant adipose tissue. The physiological significance and possible basis for these species differences in adipose metabolism are discussed.     

PINOCYTOSIS IN FIBROBLASTS : Quantitative Studies In Vitro

Horseradish peroxidase (HRP) was used as a marker to determine the rate of ongoing pinocytosis in several fibroblast cell lines. The enzyme was interiorized in the fluid phase without evidence of adsorption to the cell surface. Cytochemical reaction product was not found on the cell surface and was visualized only within intracellular vesicles and granules. Uptake was directly proportional to the administered concentration of HRP and to the duration of exposure. The rate of HRP uptake was 0.0032–0.0035% of the administered load per 10⁶ cells per hour for all cells studied with one exception: L cells, after reaching confluence, progressively increased their pinocytic activity two- to fourfold. After uptake of HRP, L cells inactivated HRP with a half-life of 6–8 h. Certain metabolic requirements of pinocytosis were then studied in detail in L cells. Raising the environmental temperature increased pinocytosis over a range of 2–38°C. The Q₁₀ was 2.7 and the activation energy, 17.6 kcal/mol. Studies on the levels of cellular ATP in the presence of various metabolic inhibitors (fluoride, 2-desoxyglycose, azide, and cyanide) showed that L cells synthesized ATP by both glycolytic and respiratory pathways. A combination of a glycolytic and a respiratory inhibitor was needed to depress cellular ATP levels as well as pinocytic activity to 10–20% of control values, whereas drugs administered individually had only partial effects. In spite of the availability of an accurate quantitative assay for fluid and solute uptake, the function of pinocytosis in tissue culture cells remains unknown.     

Increased postprandial fatty acid trapping in subcutaneous adipose tissue in obese women

The objective of this study was to test the hypothesis that increased fatty acid trapping by subcutaneous adipose tissue might contribute to the development and/or maintenance of obesity. To do so, venoarterial (V-A) gradients across subcutaneous adipose tissue for triglycerides, glycerol, nonesterified fatty acid (NEFA), and acylation-stimulating protein (ASP) were determined in eight lean females [body mass index (BMI), 22.2 +/- 0.6] and eight obese females (BMI, 34.4 +/- 3.4). Plasma insulin was also measured at intervals throughout this period. Fasting plasma triglyceride was significantly higher in the obese group and postprandial triglyceride was also significantly delayed. In contrast, both triglyceride clearance and fatty acid uptake by subcutaneous adipose tissue were significantly greater in the obese group compared with the lean group. Fasting insulin did not differ between the groups, but postprandial insulin values were significantly higher in the obese group. The pattern of ASP release from subcutaneous adipose tissue also appeared to differ in that it was significantly greater in the early postprandial period (0;-90 min) in the obese group versus the lean group and this correlated with greater triglyceride clearance during this period. Moreover, there were strong, positive correlations between BMI and the V-A gradient for fasting ASP, the 0- to 90-min area under the curve (AUC) for ASP V-A gradient fasting insulin, and the 0- to 90-min AUC for fatty acid incorporation into adipose tissue. Taken together, these data demonstrate that fatty acid trapping by adipose tissue can be increased even when overall plasma triglyceride clearance is delayed. The postprandial pattern of insulin, in particular, was altered in the obese, although it is certainly possible that differences in ASP release or response could also contribute to increased fatty acid trapping in the obese.The data, therefore, suggest that increased fatty acid trapping by adipose tissue may be a feature of some forms of obesity.     

Defective Adipose Lipolysis and Altered Global Energy Metabolism in Mice with Adipose Overexpression of the Lipolytic Inhibitor G0/G1 Switch Gene 2 (G0S2)

Biochemical and cell-based studies have identified the G0S2 (G₀/G₁ switch gene 2) as a selective inhibitor of the key intracellular triacylglycerol hydrolase, adipose triglyceride lipase. To better understand the physiological role of G0S2, we constructed an adipose tissue-specific G0S2 transgenic mouse model. In comparison with wild type animals, the transgenic mice exhibited a significant increase in overall fat mass and a decrease in peripheral triglyceride accumulation. Basal and adrenergically stimulated lipolysis was attenuated in adipose explants isolated from the transgenic mice. Following fasting or injection of a β3-adrenergic agonist, in vivo lipolysis and ketogenesis were decreased in G0S2 transgenic mice when compared with wild type animals. Consequently, adipose overexpression of G0S2 prevented the “switch” of energy substrate from carbohydrates to fatty acids during fasting. Moreover, G0S2 overexpression promoted accumulation of more and larger lipid droplets in brown adipocytes without impacting either mitochondrial morphology or expression of oxidative genes. This phenotypic change was accompanied by defective cold adaptation. Furthermore, feeding with a high fat diet caused a greater gain of both body weight and adiposity in the transgenic mice. The transgenic mice also displayed a decrease in fasting plasma levels of free fatty acid, triglyceride, and insulin as well as improved glucose and insulin tolerance. Cumulatively, these results indicate that fat-specific G0S2 overexpression uncouples adiposity from insulin sensitivity and overall metabolic health through inhibiting adipose lipolysis and decreasing circulating fatty acids.     

Analysis of the pinocytic process in rat kidney II. Biochemical composition of pinocytic vesicles compared to brush border microvilli,lysosomes and basolateral plasma membranes

Pinocytic vesicles, brush border microvilli, lysosomes and basolateral plasma membranes were isolated from rat kidney cortex and their biochemical composition and membrane turnover compared. Pinocytic vesicles are devoid of marker enzymes of brush border microvilli, such as alkaline phosphatase and 5′-nucleotidase, and of lysosomes, such as acid phosphatase and β-glucuronidase. The protein pattern as revealed by polyacrylamide gel electrophoresis differs for all four membranes. Analysis of the phospholipid composition shows that pinocytic vesicles are rich in the negatively charged phospholipid phosphatidylserine and have a low content of sphingomyelin and phosphatidylethanolamine.[¹⁴C]guanido-arginine, [³H]fucose and $\text{myo-}\text{[}{}^3\text{H]inositol}$ were preferentially incorporated into the pinocytic vesicles. Using a double label technique with leucine also, evidence of a more rapid turnover of the pinocytic vesicle membrane proteins was obtained.The results suggest that pinocytic vesicles are not derived from the brush border microvillous membrane but are independent entities that are newly synthesized during the pinocytic process.     

Distribution of radioactive glycerol and fatty acids among adipose tissue triglycerides after administration of glucose-U-14C

Adipose lipid obtained from fed rats 15 or 60 min after injection of radioactive glucose was separated into 10 triglyceride classes of differing fatty acid compositions. The distribution among these classes of total and radioactive triglyceride-glycerol was determined and found to be the same. Thus newly synthesized adipose triglycerides resemble in kind and proportion the triglycerides which exist in the tissue. This finding is in accord with the concept that the structures of adipose triglycerides are stable over long periods and that the turnover rate of the several triglyceride species are similar. After administration of radioactive glucose, the specific activity of saturated fatty acids was higher in the more saturated triglyceride species. These data indicate that newly formed saturated acids do not mix completely with all adipose tissue fatty acids available for esterification. Fatty acids derived from plasma triglyceride influenced the composition of newly synthesized adipose tissue triglyceride and thus constitute an important source of adipose tissue lipid.