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 (14)CO(2) from U-glucose-(14)C 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 (14)CO(2) 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.

Structure-function relationships in the adipose cell. II. Pinocytosis and factors influencing its activity in the isolated adipose cell.

Pinocytic activity in the adipose cell has been examined by measuring the uptake of colloidal gold. Pinocytic activity occurs in the isolated adipose cell under all experimental conditions; a portion of the vesicular elements of the cell can be identified by electron microscopy as pinocytic in origin. The isolated adipose cell appears to take up serum albumin by pinocytosis. Pinocytic activity in the isolated adipose cell is enhanced by epinephrine, but not by insulin. The relationship between pinocytosis and the metabolic activity of the adipose cell has been studied by measuring simultaneously the uptake of radioactive colloidal gold, the incorporation of (14)C-counts from U-glucose-(14)C into CO(2), total lipid, triglyceride glycerol and triglyceride fatty acids, and the release of nonesterified fatty acids in the absence of hormones and in the presence of insulin or epinephrine. Correlations between hormone-produced alterations in lipid metabolism and in pinocytic activity suggest that intracellular nonesterified fatty acid levels are a factor in the regulation of both the cell's pinocytic activity and its metabolism and that pinocytosis in the adipose cell functions in the extracellular-intracellular transport of nonesterified fatty acids.

Structure-function relationships in the adipose cell. 3. 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-(14)C and the incorporation of (14)C-counts into triglyceride glycerol and inhibits the incorporation of (14)C-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.

Changes in glucose oxidation during growth of embryonic heart cells in culture.

A rapid and convenient method has been utilized to investigate glucose oxidation during growth of chick embryo heart cells in tissue culture. Primary isolates of chick embryo heart cells showed exponential growth when plated at low densities and exhibited density-inhibited growth as cultures became confluent. The density-dependent growth inhibition of chick embryo heart cells is associated with a marked decrease in the specific activity of glucose oxidation to CO(2). This decrease in glucose oxidation was observed as density increased as either a function of time in culture or as related to initial plating density. The decrease in (14)CO(2) production associated with density-dependent inhibition of growth is due to a marked decrease in activity of the pentose phosphate pathway.

The fine structure, potassium content, and respiratory activity of isolated rat liver parenchymal cells prepared by improved enzymatic techniques.

Further modifications of the enzymatic technique for the preparation of isolated, intact, parenchymal cells from rat liver as previously described by this laboratory are presented together with a detailed account of several critical factors involved during the procedure. In addition, the fine structure of the cells as revealed by electron microscopy and the characteristics of their respiratory activity in different media and with several added substrates are described. It is shown that cells obtained by adding calcium during the preparative procedure retain approximately 34% more potassium than cells prepared solely in a calcium-free medium. The former cells also demonstrate a higher respiratory activity, which is not due to uncoupling of respiration. Electron microscopy reveals that the cells have an intact plasma membrane and well-preserved intracellular organelles. Glycogen particles are observed in all cells and are particularly abundant when either 20 mM pyruvate is added during the preparation or Eagle's Minimum Essential Medium is employed.

Thiocarbamate sulfoxides: potent, selective, and biodegradable herbicides.

Sulfoxidation of thiocarbamates yields a new class of chemicals having increased herbicidal activity along with greater tolerance of corn and soybeans in greenhouse tests. However, their thermal stability is not favorable. These sulfoxides are intermediates in the mammalian metabolism of thiocarbamates, being formed by liver microsomal oxidases and cleaved in a system consisting of glutathione and a soluble enzyme from liver.

Cholesterol: vitamin C controls its transformation to bile acids.

Cholesterol accumulates in the blood serum and in the liver of guinea pigs with chronic latent vitamin C deficiency. The reason for this is the decreased rate of transformation of cholesterol to bile acids in the liver of animals deficient in vitamin C. A significant direct correlation exists between the vitamin C concentration in the liver and the rate of cholesterol transformation to bile acids.

Detection of inborn errors of metabolism: galactosemia.

Radioautography of cultured, human, galactosemic and nongalactosemic cells shows that, in the presence of 0.05M D-galactono-gamma-lactone, the former incorporate much less galactose in acid-insoluble form than the latter. Presumably the lactone inhibits incorporation of the labeled galactose into pathways which do not require galactose-1-phosphate uridylyltransferase activity. Definite differences between the galactosemic and nongalactosemic condition can be demonstrated with as few as 100 to 1000 cells. This approach may be useful in facilitating prenatal detection of several kinds of inborn errors of metabolism.

Linoleic acid hydroperoxide: impaired bacterial uptake by alveolar macrophages, a mechanism of oxidant lung injury.

Exogenous linoleic acid hydroperoxide causes in vitro impairment of both bacterial uptake and the phagocytic stimulation of (14)CO(2) production from [1-(14)C]glucose in rabbit alveolar macrophages by an undefined effect on the cell membrane. This effect may be one mechanism for the defective pulmonary bacterial clearance characteristic of oxidant lung injury.

Difference in hepatic metabolism of long- and medium-chain fatty acids: the role of fatty acid chain length in the production of the alcoholic fatty liver.

Replacement of dietary triglycerides containing long-chain fatty acids (LCFA) by triglycerides containing medium-chain fatty acids (MCFA) markedly reduced the capacity of alcohol to produce fatty liver in rats. After 24 days of ethanol and MCFA, the increase in hepatic triglycerides was only 3 times that of controls, whereas an 8-fold rise was observed after ethanol and LCFA. The triglyceride fatty acids that accumulated in the liver after feeding of ethanol with MCFA contained only a small percentage of the MCFA; their composition also differed strikingly from that of adipose lipids. To study the mechanism of the reduction in steatosis, we compared oxidation to CO(2) and incorporation into esterified lipids of (14)C-labeled chylomicrons or palmitate-(14)C (representing LCFA), and of octanoate-(14)C (as MCFA) in liver slices and isolated perfused livers, in the presence or absence of ethanol. Ethanol depressed the oxidation of all substrates to CO(2); MCFA, however, was much more oxidized and reciprocally much less esterified than LCFA, with a 100-fold difference in the ratio of esterified lipid-(14)C to (14)CO(2). Furthermore, in hepatic microsomal fractions incubated with alpha-glycerophosphate, octanoate was much less esterified than palmitate. This propensity of MCFA to oxidation rather than esterification represents a likely explanation for the reduction in alcoholic steatosis upon replacement of dietary LCFA by MCFA.

The reduction of glyceraldehyde by human erythrocytes. L-hexonate dehydrogenase activity.

Incubation of red cell suspensions with D-glyceraldehyde resulted in disappearance of glyceraldehyde and appearance of glycerol. Concomitantly, there was an increase of CO(2) formation from glucose. This indicated that the reduction of glyceraldehyde to glycerol occurred through a NADPH-linked system. Studies in hemolysates revealed the presence of an enzyme with the capacity to catalyze the reduction of glyceraldehyde to glycerol by NADPH. This enzyme was partially purified by DEAE chromatography. The elution pattern of the enzyme and its kinetic characteristics indicated that the enzyme was L-hexonate dehydrogenase (L-gulonate: NADP oxidoreductase, EC 1.1.1.19), not aldose reductase (Alditol: NADP oxidoreductase, EC 1.1.1.21), which had previously been thought present in erythrocytes. The reduction of glyceraldehyde to glycerol is one of a number of pathways for the metabolism of glyceraldehyde that have been found in red cells and/or other mammalian tissues.

Cellular basis of insulin insensitivity in large rat adipocytes.

The marked stimulatory effect of insulin on the conversion of 20 mM D-[6-14C]glucose to CO2, glyceride-glycerol, and fatty acid observed in small rat adipocytes was greatly diminished in large cells from older rats. Similarly, total glucose utilization as estimated by summing the total metabolites accumulated intracellularly plus the release of labeled CO2 and lactate was substantially lower in large cells in the presence of insulin and 5 mM labeled glucose. However, under conditions of 0.2 mM medium glucose where transport of the hexose into adipocytes is relatively more rate-limiting for subsequent metabolism, large cells actually utilized slightly greater total amounts of glucose than small cells in the presence of insulin. Increments of total glucose utilization due to both submaximal and maximal doses of insulin were similar in large and small cells incubated with a low glucose concentration. Under these conditions, conversion of labeled glucose to CO2 and fatty acid in response to insulin was somewhat diminished in large cells, while conversion to glyceride-glycerol was enhanced. The activity of the D-glucose transport system in large and small cells was estimated by monitoring initial rates and small cells was estimated by monitoring initial rates of 3-O-[3H]methylglucose uptake by a rapid filtration method. Transport system activity on a per cell basis was actually severalfold higher in large adipocytes in the basal state as well as in the presence of submaximal and maximal concentrations of insulin compared to small cells. However, the percent stimulation by insulin was less in the large cells. Uptake of 2-deoxyglucose under basal conditions and in response to insulin was also higher in large cells compared to small cells. Analysis of the accumulated label in extracts from fat cells incubated with D-[14C]deoxyglucose revealed the presence of free deoxyglucose, deoxyglucose-6-phosphate, and 6-phosphodeoxygluconate. The levels of these metabolites were significantly higher in large cells compared to small cells indicating hexokinase activity appears not to account for the defective glucose utilization in large cells at high glucose concentrations. It is concluded that (a) possible defects in insulin receptor components, the D-glucose transport system, and the coupling mechanism which links these entities do not significantly contribute to the apparent insulin-insensitivity of large fat cells and (b) the principal cellular defect which confers this blunted insulin response to large rat adipocytes involves one or more intracellular enzymes involved in glucose metabolism.

An isotopic assay for NADPH oxidase activity and some characteristics of the enzyme from human polymorphonuclear leukocytes.

An isotopic assay for NADPH ixodase that measures the amount of NADP formed by the 6-phosphogluconate dehydrogenase reaction has been developed. Under appropriate conditions, the amount of NADP present is directly proportional to the amount of 14CO2 released from [1-14C]6-phosphogluconic acid. Because this assay employs radioisotopes, it is far more sensitive than conventional assays for the enzyme. The human granule NADPH oxidase, as measured by this assay, is active in the presence of CN minus, is stimulated by Mn-2+, and has a pth optimum of 5.5. Granules isolated from cells that have been allowed to ingest zymosan consistently exhibited more enzyme activity than did granules isolated from either resting cells or cells challenged with zymosan that was not preopsonized. This effect was observed over a wide range of substrate concentrations and could not be explained by differences in protein concentrations between the various samples. If whole homogenates are used in place of isolated granules, the enzyme activity can be observed only with a homogenate of phagocytizing cells and even then only at a high concentration of NADPH. This suggests that an inhibitor of the enzyme might be present within the cell. One patient with chronic granulomatous disease was studied. There was no difference in tnadph oxidase activity of the patients' cells when granules from resting and phagocytizing cells were compared. In contrast, the enzyme activity in granules from two control patients doubled upon phagocytosis. These results are consistent with a role for NADPH oxidase in the initiation of the respiratory burst accompanying phagocytosis by human neutrophils.

The biological and immunological properties of pork and beef insulin, proinsulin, and connecting peptides.

The recently discovered hormone precursors, pork and beef proinsulins, their respective connecting peptides, and beef proinsulin intermediates have been compared to insulin in their ability to stimulate the conversion of glucose-U-(14)C to (14)CO(2) and lipids in isolated fat cells. The concentrations of beef and pork proinsulins required to achieve the same biological effect were respectively, 15 and 10 times that of insulin. Beef proinsulin intermediates required only 2.6 times the concentration of insulin for the same effect. Pork and beef connecting peptides in high or low concentrations alone or in combination with proinsulin, insulin, or proinsulin intermediates showed no biological effect on the isolated fat cell system. The insulin-like activity of beef and pork proinsulins on the isolated fat cell system was not abolished with pancreatic trypsin or kallikrein inhibitors. Pork insulin antiserum inhibited the biological activity of pork insulin and proinsulin as well as that of beef insulin or proinsulin. Pork proinsulin antiserum also inhibited the insulin-like activity of both pork insulin and proinsulin. By the radioimmunoassay method, pork insulin antiserum bound only (1/4) to [unk] as much proinsulin as insulin. Beef proinsulin intermediates, on the other hand, were found to react with the pork insulin antiserum to an extent nearly equal to that of insulin. These data suggest that (a) proinsulin exhibits its effect on the isolated fat cells independent of its conversion to insulin, (b) connecting peptides have no biological effect under present experimental conditions, and (c) in comparison to insulin, immunological reactivity of proinsulin is greater than its biological activity using our pork insulin antiserum; thus, the comparison of antibody specificity with the fat cell receptor specificity suggests that the biological site of action is different from the immunologic site.

Free fatty acid metabolism of leg muscles during exercise in patients with obliterative iliac and femoral artery disease before and after reconstructive surgery.

The free fatty acid (FFA) uptake and oxidation and the carbohydrate substrate exchange of leg muscles were studied during exercise in 14 patients with occlusive disease of the iliac or femoral arteries before and 3-6 months after reconstructive vascular surgery and in 5 healthy subjects. (14)C-labeled oleic acid was infused continuously at rest and during exercise at work loads of 150-400 kg-m/min. The arterial concentration of FFA was similar both at rest and during exercise in patients and controls. The patients showed a smaller increase in the fractional turnover of FFA during exercise. Leg uptake and release of FFA in terms of micromoles per liter plasma did not differ significantly either at rest or during exercise between patients and controls. FFA oxidation could not be measured at rest but exercise data showed a lower fractional oxidation of FFA (P < 0.001) in the patient group (53+/-6%) compared with the controls (84+/-2%). For the entire material, fractional oxidation of FFA showed a significant negative regression on the lactate/pyruvate ratio in femoral venous blood. The ventilatory respiratory quotient (RQ) and the leg muscle exchange of glucose and lactate in the patients exceeded that of the controls. When six patients were studied after reconstructive surgery, fractional oxidation of FFA had risen from a preoperative value of 47+/-8 to 90+/-10%, other data for leg muscle FFA metabolism being unchanged. IT IS CONCLUDED: (a) that substrate catabolism by the leg muscles during exercise in these patients proceeds in excess of the simultaneous capacity to oxidize acetyl-CoA in the tricarboxylic acid cycle, and (b) oxidation of FFA by contracting muscle is related to the muscle cell redox state.

Pathways of glutamine and organic acid metabolism in renal cortex in chronic metabolic acidosis.

The metabolism of labeled glutamine and of several labeled organic acid anions was compared in tissue slices of renal cortex from chronically acidotic and alkalotic littermate dogs. (15)NH(3) formation and (15)N-amideglutamine utilization were significantly increased by slices from acidotic animals providing further evidence for the similarity of the metabolic responses seen in the tissue slice system and the physiologic effects produced by chronic metabolic acidosis on renal metabolism in the intact animal. Slices from acidotic dogs formed more (14)CO(2) and glucose-(14)C than did slices from alkalotic animals when labeled glutamine, citrate, or malate was used as substrate but (14)CO(2) production from pyruvate-1-(14)C was slightly reduced in acidotic tissue. With most of the substrates used glucose-(14)C formation was small compared with (14)CO(2) formation. Using the amount of glucose-(14)C formed, the expected (14)CO(2) production was calculated based on the hypothesis that the primary site of action of metabolic acidosis is on a cytoplasmic step in gluconeogenesis. The actual difference in (14)CO(2) production between slices from acidotic and alkalotic animals always greatly exceeded this predicted amount, indicating that stimulation of gluconeogenesis represents a minor metabolic response to chronic metabolic acidosis. Evidence from experiments with citrate labeled in various positions showed that metabolic acidosis has its principal effect on an early step in substrate metabolism which must be intramitochondrial in location.

Catalase-dependent peroxidative metabolism in the alveolar macrophage during phagocytosis.

EVIDENCE FOR THE PRESENCE OF PEROXIDATIVE METABOLISM IN RABBIT ALVEOLAR MACROPHAGES (AM) HAS BEEN OBTAINED FROM THE FOLLOWING OBSERVATIONS: (a) catalase is present in high concentrations; (b) peroxidase activity could not be detected employing guaiacol as substrate; (c) the irreversible inhibition of AM catalase by aminotriazole served as a detection system for H(2)O(2) and demonstrated increased intracellular H(2)O(2) after phagocytosis; (d) formate oxidation, a marker of catalase-dependent peroxidations, occurs in resting AM and is increased by phagocytosis; (c) measurements of H(2)O(2) accumulation in a dialysate of AM demonstrated twofold increase during phagocytosis; and (f) aminotriazole diminishes O(2) utilization and (14)CO(2) production from labelled glucose and pyruvate. It is concluded that, while catalase-dependent H(2)O(2) metabolism is not essential for particle entry, this pathway represents one of the metabolic pathways stimulated by particle entry in the AM.

Metabolism of [1-(14)C] and [2-(14)C] leucine in cultured skin fibroblasts from patients with isovaleric acidemia. Characterization of metabolic defects.

Leucine metabolism in cultured skin fibroblasts from patients with isovaleric acidemia was compared with that in normal fibroblasts and in cells from patients with maple syrup urine disease using [1-(14)C] and [2-(14)C] leucine as substrates. Inhibitory effects of methylenecyclopropylacetic acid on leucine metabolism in normal cells were also investigated. Production of 14CO2 from [2-(14)C] leucine was very reduced (96-99%) in both types of mutant cells. Radioactive isovaleric acid accumulated in assay media with isovaleric acidemia cells but not in those with maple syrup urine disease cells. Unexpectedly, 14CO2 production from [1-(14)C] leucine was partially depressed (80%) in isovaleric acidemia cells whereas in maple syrup urine disease cells it was strongly depressed (99%) as expected. These two mutant cells were clearly distinguished by detection of 14C-isovaleric acid accumulation after incubation with [2-(14)C] leucine. A pattern of inhibition of leucine oxidation similar to that seen in isovaleric acidemia cells was induced in normal cells by the addition of 0.7 mM methylenecyclopropylacetic acid to the assay medium. The partial inhibition of [1-(14)C] leucine oxidation seen in isovaleric acidemia cells and also in normal cells in the presence of the inhibitor appears to be, at least in part, due to an accumulation of isovalerate in the cells. Isovaleric acid (5-10) mM) inhibited [1-(14)C] leucine oxidation 32-68% when added to the assay medium with normal cells. Addition of flavin adenine dinucleoside to culture medium or assay medium or both did not restore oxidation of either leucine substrate in isovaleric acidemia cells.

Utilization of alpha-keto and alpha-hydroxy analogues of valine by the growing rat.

When 70-80-g male albino rats eat a diet furnishing daily requirement of valine for optimal growth (70 mumol/g) and all other nutrients ("complete diet"), they gain weight at an average rate of 3.0 g/100 g body wt/day. When valine is removed, they lose weight at an average 2.1 g/100 g body wt/day. The growth retardation is improved or corrected by adding valine to the diet, daily weight gain being proportional to dietary valine content over a range of 0-70 mumol/g. Addition of alpha-ketoisovaleric acid instead of valine to the valine-free diet also improves or corrects the growth failure. Percent efficiency of alpha-ketoisovaleric acid as a substitute for valine was calculated as: 100 x (micromole valine per gram diet required to produce specified growth response)/(micromole alpha-ketoisovaleric acid per gram diet required to produce the same response). Efficiency of the substitution is inversely related to dietary content of the keto analogue, being 80% when diet contains 17.5 mumol/g (molar equivalent of (1/4) the daily requirement of valine), and 37% when diet provides 140 mumol/g (molar equivalent of twice the daily requirement of valine).alpha-Hydroxyisovaleric acid also substitutes for valine. Efficiency of the substitution at the single ration tested, 70 mumol/g diet, is 45%, similar to that for the keto analogue under the same conditions. When [1-(14)C]alpha-ketoisovaleric acid is injected intravenously, 30-80% of the administered radioactivity is exhaled as (14)CO(2) within 24 h. This finding suggests that inefficiency of alpha-ketoisovaleric acid as a substitute for valine results in part from degradation of the keto acid to isobutyric acid by branched chain dehydrogenase-decarboxylase. Oral administration of neomycin, polymyxin, and bacitracin reduces efficiency of alpha-ketoisovaleric acid as a substitute for valine by (1/4)-(1/2). This effect suggests that transamination of the keto acid may be performed in part by gastrointestinal microbes.

Erythrocyte catabolism by macrophages in vitro. The effect of hydrocortisone on erythrophagocytosis and on the induction of heme oxygenase.

Phagocytosis of erythrocytes was studied in vitro in an incubation system consisting of rat peritoneal macrophages and antibody-coated (59)Fe-labeled erythrocytes. The system was characterized in terms of the rate and magnitude of erythrophagocytosis, determined by the interiorization of the (59)Fe label. On incubation of 150 x 10(6) macrophages with 75 x 10(6) antibodycoated erythrocytes, erythrophagocytosis began within a few minutes and was essentially completed after 2 h when 50% of the offered red cells had been ingested by the macrophages. Heme oxygenase (HO) activity, which is very low in native macrophages, increased 4- to 10- fold in response to the ingested erythrocytes; this enzyme stimulation occurred with a delay of 3 h in relation to erythrophagocytosis. Actinomycin D or puromycin prevented the increase of HO activity without affecting erythrophagocytosis, which suggests that the enzyme stimulation was due to substrate-mediated enzyme induction. Hydrocortisone (HC) (0.1 mg/ml medium) dissociated erythrophagocytosis from HO induction, leaving the former unimpaired but completely suppressing the latter. The suppressive effect of HC on the enzyme induction was completely prevented by 5 mg glucose and 0.02 U insulin/ml of the medium. In macrophages engaged in erythrophagocytosis. HC also lowered glucose removal from the medium and reduced formation of (14)CO(2) from [1-(14)C]glucose. These results suggest that induction of HO in macrophages by the hemoglobin of ingested erythrocytes requires intact transport or metabolism of glucose. Glucose utilization appears to be impaired by HC, but is restored by additional glucose and insulin. The findings suggest that plasma steroid concentrations in the pharmacological range could reduce bilirubin formation in phagocytic cells in vivo without affecting the sequestration and degradation of erythrocytes. This provides a possible explanation for the observation that in patients with hepatogenous jaundice, steroids often lower the serum bilirubin level.