Expression of the third member of the serum amyloid A gene family in mouse adipocytes.

Three homologous genes that code for three related proteins comprise the serum amyloid A (SAA) family in the mouse. Endotoxin induces equally vigorous expression of mRNAs for the three SAA genes in liver. In extrahepatic tissues SAA1 and/or SAA2 mRNAs have been found only in kidney and intestine, however, SAA3 is expressed in all extrahepatic tissues thus far examined. This observation raised the question: is SAA3 mRNA expressed by a single cell system dispersed throughout all tissues, or by differentiated cells of each tissue? This question was explored in various tissues by in situ hybridization with a single-stranded cRNA probe specific for SAA3 mRNA. We found expression in the liver of SAA3 mRNA by other cells as well as by hepatocytes. A common feature among extrahepatic tissues was SAA3 mRNA expression in adipocytes. SAA3 mRNA was also found in two nonadipose cells, Leydig cells of the testis, and some of the cells located in parafollicular zones of the spleen.

Lipid synthesis, intracellular transport, storage, and secretion. I. Electron microscopic radioautographic study of liver after injection of tritiated palmitate or glycerol in fasted and ethanol-treated rats.

A time sequence study of intracellular movement of labeled lipid in the liver was carried out on fasted and ethanol-treated rats injected with either palmitate-(3)H or glycerol-(3)H by electron microscopic radioautography. The elimination of water-soluble lipid precursors during specimen preparation was checked and found to be complete. The labeled lipid product in the tissue was identified as mostly triglyceride. A dehydration procedure was adapted to minimize the loss of lipid during specimen preparation. At 2 min after injection, the earliest time interval studied, both precursors were found to have penetrated the liver cells, and the label was found over both rough and smooth elements of the endoplasmic reticulum, which is the site of glyceride esterification. From 5 min on, in fasted and especially in ethanol-treated rats, the label was seen also over lipid droplets 0.5-2.0 micro in diameter, which represent "storage lipid" (slowly turning over compartment). Mitochondria became labeled mostly at later time intervals after injection. From 10 min on, concentration of label was seen over the Golgi apparatus, containing small osmiophilic particles. Association of label with groups of particles in smooth-surfaced vesicles and vacuoles in and near the Golgi apparatus and in the vicinity of the sinusoidal border was seen, both after palmitate-(3)H and glycerol-(3)H. It is proposed that these particles represent lipoproteins which are formed in the endoplasmic reticulum, "processed" in the Golgi apparatus, and transported in vacuoles to the sinusoid surface to be discharged into the circulation.

Decline of DDT residues in migratory songbirds.

Analyses of ten species of migratory songbirds killed when the birds flew into television towers in Florida showed a progressive decline in the concentration of DDT and its metabolites (DDD and DDE) in their fat depots for the period 1964 to 1973. This decline is apparently correlated with the decreased usage of DDT in the United States during the same time.

Nonconversion of o,p'-DDT to p,p'-DDT in rats, sheep, chickens, and quail.

The finding of appreciable quantities of p,p'-DDT after feeding o,p'-DDT to rats led to the proposal of a theory, that an isomeric metabolic conversion occurs. The presence of p,p'-DDT as an impurity in supposedly pure samples of o,p'-DDT is the correct explanation for the appearance of p,p'-DDT. Purified o,p'-DDT and (14)C-labeled o,p'-DDT yielded no data to support the idea that o,p'-DDT is converted to the p,p'-DDT isomer.

Oxychlordane, animal metabolite of chlordane: isolation and synthesis.

Oxychlordane (C(10)H(4)Cl(8)O), a minor heretofore unidentified metabolite, was isolated from fat of pigs on diets heavily dosed with pure isomers of the insecticide, chlordane (C(10)H(6)Cl(8)). Chemical and spectroscopic evidence provides bases for proposal of structure of the metabolite as 1-exo-2-endo-4,5,6,7,8,8-octachloro-2,3-exo-epoxy-2,3,3a,4,7,7a-hexahydro-4,7-methanoindene.

Rats enriched with odd-carbon fatty acids: maintenance of liver glycogen during starvation.

In young rats a diet containing triundecanoin as the major source of fat produces substantial enrichment of adipose tissue triglycerides with undecanoate and higher fatty acids with odd-numbered carbons. The terminal three-carbon residues arising from beta-oxidation of these acids are glucogenic and help to counteract the decreases in liver glycogen and serum glucose ordinarily induced by prolonged fasting.

Natural abundance carbon-13 nuclear magnetic resonance spectra of the canine sciatic nerve.

The proton-decoupled natural abundance carbon-13 nuclear magnetic resonance spectrum of the canine sciatic nerve is virtually identical to that of canine adipose tissue and markedly similar to that of liquid triolein. No resonances assignable to cholesterol, glycolipids, or sphingolipids are detectable in the sciatic nerve spectrum despite their abundance in the myelin sheath of this nerve. However, many such resonances are observed in lipid extracts of the nerve. Chronmatographic analysis of specimens of canine and rabbit sciatic nerve has revealed that these contain sufficient triglyceride to account quantitatively for the observed spectrum. Proton nuclear magnetic resonance and spin-labeling results for preparations containing myelin, especially those derived from the peripheral nerve, should be critically examined for experimental artifacts reflecting the triglyceride content.

Lipolysis in homogenates of adipose tissue: an inhibitor found in fat from obese rats.

The presence of a lipidbound inhibitor in adipose tissue of rats with hypothalamic obesity may explain the failure of the tissue to release fatty acids on epinephrine stinmulation. Aqueous extracts of tissue from obese animals showed no deficiency of lipase activity, but when whole homogenates of epididymal fat from lean and obese animals were mixed, 25 percent tissue from obese animals reduced by 73 percent the release expected from tissue of lean controls.

Plasticizers from plastic devices extraction, metabolism, and accumulation by biological systems.

Phthalate ester plasticizers were found to be extracted by blood from plastic tubing and from plastic bags used for blood storage. One such plasticizer was metabolized by the isolated perfused rat liver while another was found to be accumulated in the liver unchanged. In addition, this latter plasticizer was identified in samples of human tissue taken from patients who had received transfusions of blood stored in plastic bags. The biological implications of these observations are considered.

DDT administered to neonatal rats induces persistent estrus syndrome.

The o,p'-isomer of the insecticide DDT when injected into neonatal female rats significantly advanced puberty, induced persistent vaginal estrus after a period of normal estrous cycles, and caused the ovaries to develop follicular cysts and a reduced number of corpora lutea. The uterotropic response to administered estradiol was reduced, and the female pattern of mating behavior was slightly disturbed. Residues of DDT in ovarian, brain, and adipose tissues of the adult animals were the same in both treated and control groups.

Adrenergic regulation of lipolysis in situ at rest and during exercise.

The adrenergic regulation of lipolysis was investigated in situ at rest and during standardized bicycle exercise in nonobese healthy subjects, using microdialysis of the extracellular space in subcutaneous adipose tissue. The glycerol concentration was about two times greater in adipose tissue than in venous blood. At rest, the glycerol concentration in adipose tissue was rapidly increased by 100% (P less than 0.01) after the addition of phentolamine to the ingoing perfusate, whereas addition of propranolol did not alter the adipose tissue glycerol level. Glycerol in adipose tissue and plasma increased during exercise and decreased in the postexercise period. Propranolol in the perfusate almost completely inhibited the increase in the tissue dialysate glycerol during the exercise-postexercise period. Phentolamine, however, was completely ineffective in this respect. During exercise, the lipolytic activity was significantly more marked in abdominal than in gluteal adipose tissue; this was much more apparent in women than in men. Thus, in vivo lipolysis in subcutaneous adipose tissue is regulated by different adrenergic mechanisms at rest and during exercise. Alpha-adrenergic inhibitory effects modulate lipolysis at rest, whereas beta-adrenergic stimulatory effects modulate lipolysis during exercise. In addition, regional differences in lipolysis are present in vivo during exercise, which seem governed by factors relating to sex.

Deposition in and release of vitamin D3 from body fat: evidence for a storage site in the rat.

Vitamin D in all body tissues was radio-labeled by supplementing completely vitamin D-deficient weanling rats with oral vitamin D(3)-4-(14)C for 2 wk. All vitamin D was then withheld, and radioactivity and vitamin D content in a variety of organs and tissues were measured. Adipose tissue was found to contain by far the greatest quantity of radioactivity throughout the 3 month experimental period. Immediately after supplementation, half of the total radioactivity in adipose tissue corresponded to unaltered vitamin D(3), and the other half to polar metabolites and esters of vitamin D(3) and unidentified peak II. 1 month later there was approximately the same proportion but a decrease in the total quantity of each form. We conclude that adipose tissue is the major storage site for vitamin D(3) in its several forms. Unaltered vitamin D(3) was the principal storage form observed and presumably a source available for conversion to other metabolites during deprivation.

Carbohydrate metabolism in pregnancy. 8. Metabolism of adipose tissue isolated from fed and fasted pregnant rats during late gestation.

The effects of late pregnancy on adipose tissue metabolism have been examined in fed and fasted rats. Lumbar fat was excised from 19-day pregnant and age-matched virgin rats which had been given unrestricted access to food ("fed") or fasted for 48 hr before sacrifice. In the fed state, adipose tissue from pregnant rats displayed an increased content of free fatty acids (FFA). This coincided with augmented cleavage of preformed glycerides during incubation in vitro as evidenced by greater net production of FFA and glycerol, and altered disposition of labeled glucose. The enhanced lipolysis was independent of the availability of glucose and was not accompanied by impaired responsiveness to the antilipolytic or to the lipogenic actions of added insulin. In the presence of glucose and albumin, esterification as well as lipolysis was greater in adipose tissue from pregnant than nongravid animals. All the differences were exaggerated by prior fasting. These properties of adipose tissue during late gestation have been ascribed to a primary activation of lipolysis rather than impaired esterification or resistance to insulin. It has been suggested that the hormones of pregnancy may be responsible. Although increased intake of food and heightened availability of insulin may offset the net lipolytic effects in the fed state, a heightened turnover of adipose stores is always present. Thus, the pregnant animal appears better poised to mobilize preformed fat whenever exogenous nutrients are withheld.

Studies of arteriosclerosis in Japanese and American men. I. Comparison of fatty acid composition of adipose tissue.

The proportions of fatty acids in lipids of subcutaneous adipose tissue was compared in closely age-matched, urban men from two populations with a great difference in mortality from arteriosclerosis, namely in 50 Americans and 56 Japanese aged 15-65 yr who had died suddenly and unexpectedly. Specimens from both groups were analyzed side by side for fatty acids by gas-liquid chromatography. Compared with Japanese, Americans had significantly (P<0.01) higher proportions of lauric (+ 0.2%), myristic (+ 0.4%), palmitic (+ 1.4%), stearic (+ 2.2%), and oleic (+ 5.3%) and lesser of palmitoleic (- 1.8%), linoleic (-6.3%), and linolenic (-0.4%) acids. Japanese had higher proportions of longer chain polyunsaturated fatty acids. The distributions of fatty acids for the groups at ages 35-44 yr had significant differences. With age, Americans showed significant increases of palmitic and oleic acids and decreases of lauric, myristic, stearic, and linoleic acids; Japanese showed no correlations of proportions of fatty acids with age. The significant correlations between per cent standard body weight and fatty acids in Americans were positive for palmitic and negative for lauric and stearic acids, and in Japanese, negative for myristic acid. The patterns of interacid correlations were dissimilar for the groups. These patterns may be stable characteristics of these groups providing further insight into their fatty acid metabolism. The relationships with the fatty acid compositions of the American and Japanese diets are discussed.

Relationship between lipoprotein lipase activity and plasma sex steroid level in obese women.

In obese women (n = 16) at their weight, fasting adipose tissue lipoprotein lipase (LPL) activity, obtained by elution with serum and heparin at 4 degrees and 37 degrees C, was inversely correlated to plasma estradiol levels (r = -0.724; P = 0.002) and (r = -0.641; P = 0.010), respectively. Furthermore, fasting postheparin plasma LPL activity during a heparin infusion, showed an even stronger inverse correlation to plasma estradiol when measured at 60 min (r = -0.815; P less than 0.001). None of the above parameters was correlated to the body mass index. Postprandial LPL activity in postheparin plasma, measured 10 min after a heparin injection, showed a strong positive correlation with plasma free testosterone (r = 0.780; P = 0.001). Neither of these parameters was correlated with the body mass index. The origin of this LPL activity is presently unknown but could conceivably represent a pool of LPL from skeletal muscle. Since it has been shown convincingly that estrogen decreases adipose tissue LPL activity in the rat, the present studies strongly suggest that estradiol is a major negative regulator of fasting adipose tissue LPL activity in women.

Beta-sitosterolemia and xanthomatosis. A newly described lipid storage disease in two sisters.

Although the usual diet may contain 150-250 mg of plant sterols, chiefly beta-sitosterol, only trace amounts of these sterols have heretofore been found in human or animal blood and tissues. We now report elevated plant sterol levels in the blood and tissues of two sisters with extensive tendon xanthomas but normal plasma cholesterol levels. Besides beta-sitosterolemia and xanthomatosis, no other physical, mental, or biochemical abnormalities were detected.Repeatedly, the plasmas of the two sisters have contained 27.1 and 17.7 mg/100 ml of beta-sitosterol, 9.7 and 8.2 mg/100 ml of campesterol, and 0.5 and 0.5 mg/100 ml of stigmasterol, respectively. These plant sterols constituted 15.6 and 11.3% of the total plasma sterols. Some 60% of the plasma beta-sitosterol and campesterol was esterified; the measurable stigmasterol was entirely unesterified. The transport of the plasma beta-sitosterol and campesterol was largely in low density lipoproteins (76 and 83%, respectively). High density lipoproteins carried the remainder. Plant sterols were barely detectable in the very low density lipoprotein fraction. Only trace amounts of stigmasterol could be detected in the low density and high density lipoprotein fractions. The plant sterol content of the red blood cells averaged 12-13 mg/100 ml packed cells or about 13% of the total sterols. Two tendon xanthoma biopsies with the usual high concentration of cholesterol had 36.7 and 4.0 mg of plant sterols/g dry wt, of which 25.7 and 2.9 mg were beta-sitosterol, entirely in the free form. Plant sterols were also found in adipose tissue (0.2 mg/g wet wt) and in skin surface lipids (3.2 mg/g of lipid). The intestinal absorption of beta-sitosterol in both the patients, measured by two techniques, indicated greatly increased absorption of this sterol (about 24 and 28% in the patients L. H. and R. H., respectively, normal absorption being <5%). We suggest that increased absorption of beta-sitosterol must be considered as one cause of this disease. The reason for the extensive xanthomatosis in these two patients remains unknown. Perhaps in some way plant sterols initiated the development of xanthomas with otherwise normal plasma cholesterol levels. Clinical atherosclerosis has not yet occurred. The occurrence of beta-sitosterolemia in these two sisters with un-affected parents suggests an inherited recessive trait.

Glucose transporter levels in spontaneously obese (db/db) insulin-resistant mice.

In the present study we examined mRNA and protein levels for the muscle/adipose tissue glucose transporter (GLUT-4) in various tissues of spontaneously obese mice (C57BL/KsJ, db/db) and their lean littermates (db/+). Obese (db/db) mice were studied at 5 wk of age, when they were rapidly gaining weight and were severely insulin resistant, evidenced by hyperglycemia (plasma glucose 683 +/- 60 vs. 169 +/- 4 mg/dl in db/+, P less than 0.05) and hyperinsulinemia (plasma insulin 14.9 +/- 0.53 vs. 1.52 +/- 0.08 ng/ml in db/+, P less than 0.05). The GLUT-4 mRNA was reduced in quadriceps muscle (67.5 +/- 8.5%, P = 0.02), but unaltered in adipose tissue (120 +/- 19%, NS), heart (95.7 +/- 6.1%, NS), or diaphragm (75.2 +/- 12.1%, NS) in obese (db/db) mice relative to levels in lean littermates. The GLUT-4 protein, measured by quantitative immunoblot analysis using two different GLUT-4 specific antibodies, was not different in five insulin-sensitive tissues including diaphragm, heart, red and white quadriceps muscle, and adipose tissue of obese (db/db) mice compared with tissue levels in lean littermates; these findings were consistent when measured relative to tissue DNA levels as an index of cell number. These data suggest that the marked defect in glucose utilization previously described in skeletal muscle of these young obese mice is not due to a decrease in the level of the major muscle glucose transporter. An alternate step in insulin-dependent activation of the glucose transport process is probably involved.

Cholesterol metabolism in human obesity.

An experiment was undertaken to test whether in severe obesity cholesterol production rates obtained by isotope kinetic analysis (two-pool compartmental analysis) are comparable to those measured by chemical sterol balance techniques. Eight severely obese but normocholesterolemic patients were studied by the balance method, and five of these eight were studied by compartmental analysis. Cholesterol turnover was 10% higher by compartmental analysis. In the entire group of eight patients cholesterol turnover was greater than twice that found previously in nonobese patients studied under similar conditions with bile acids and neutral sterols both participating in the increase. This increment was directly related to excess body fat and to adipose cellularity, with correlation co-efficients of 0.66 and 0.72, respectively. The amount of cholesterol in the slowly turning over pool B was related to degree of adiposity, but that in plasma and in pool A did not differ from values in nonobese patients.

Regulation of cholesterol metabolism in the dog. II. Effects of complete bile diversion and of cholesterol feeding on pool sizes of tissue cholesterol measured at autopsy.

In six adult pedigreed dogs the effects of high-cholesterol diets or bile diversion on the sizes of body cholesterol pools were studied at autopsy. Total body cholesterol was determined by measuring the cholesterol content of discrete organs and of the eviscerated carcass: neither cholesterol feeding nor bile diversion had altered total body cholesterol or the cholesterol content of individual organs and tissues. These results validated the conclusion based on sterol balance data obtained during life, that high-cholesterol feeding did not lead to substantial expansion of tissue cholesterol pools. THE TOTAL AMOUNT OF EXCHANGEABLE CHOLESTEROL IN THE ANIMALS WITH AN INTACT ENTEROHEPATIC CIRCULATION, WHEN ESTIMATED FROM ISOTOPIC DATA, WAS ESSENTIALLY THE SAME AS THAT MEASURED CHEMICALLY: this indicated that there was little or no nonexchangeable cholesterol in these dogs, except in skin and nervous tissue, regardless of the cholesterol content of the diet. This correspondence of estimates was not obtained in the bile-diverted dogs: we propose that the defect in the isotopic estimates was due to the accelerated rate of cholesterol synthesis in these animals. Gross and microscopic morphology of all organs and tissues was examined. Abnormal findings were limited to the biliary tract and the urinary collecting system of the two bile-diverted dogs: multiple bilirubinate gallstones were found, and mild pyelitis and ureteritis were present on the side of the bilio-renal shunt, but the urinary bladder was normal. Histologic evidence of moderate degree of cholangitis was found in one of the two bile-shunted dogs, but in neither dog was there evidence of impedance of bile flow.

Human adipocyte cholesterol. Concentration, localization, synthesis, and turnover.

By analysis of 124 specimens in 16 different patients, isolated human adipocyte cholesterol concentration is highly correlated with fat cell size but not with plasma cholesterol concentration. Less than 6 percent of total cholesterol is esterified; after subcellular fractionation, 88 percent of the cholesterol is recovered in the triglyceride-rich supernatant oil. This latter finding supports the observation that fat cell cholesterol is determined by triglyceride content, and hence by fat cell size. After intravenous administrtion of radioactive cholesterol, the sum of a three-exponential equation was fit simultaneously to both the plasma and adipocyte specific activity time curves in six patients. In five of the six, a slowly turning over pool (pool 3) closely fit the adipocyte data. Two model structures, mammillary and catenary, were fitted to the data. There was no synthesis in pool 3 using a mammillary model but a mean 5.3 percent of the total body production rate was found in compartment 3 if a catenary model was assumed. Although a catenary model is biologically unlikely, it could not be excluded. Obesity is associated with an increased cholesterol synthetic rate equal to 20 mg/day for each kilogram of body fat. To test (by an independent method) if this synthesis might be occurring in adipose tissue, human fat cells were obtained under a wide variety of dietary conditions and incubated in vitro with radioactive glucose or acetate. Incorportation of these precursors into sterol could account for no more than 1 mg cholesterol synthesis/kg fat per day. These in vitro data taken together with the in vivo mammillary compartmental analysis data are compatible with the possiblity that the excess cholesterol synthesis of obesity occurs in pool 1, most likely from hepatic or intestinal sites.