Sidney S. Chernick

Lipoprotein lipase activity of adipose and mammary tissue and plasma triglyceride in pregnant and lactating rats

Abstract 1. 1. The effect of pregnancy and lactation on lipoprotein lipase activity in adipose and mammary tissues and on plasma triglyceride concentration were studied in the rat. 2. 2. Plasma triglyceride concentration, 0.9 mM in control rats, increased 3-fold between the 12th and 20th days of pregnancy to a peak of 3.3 mM, and then decreased 50% during the next 2 days. It increased again at parturition, on the 22nd–23rd days of pregnancy, to 3.0 mM, and then fell sharply and remained below 1.0 mM throughout lactation. 3. 3. Lipoprotein lipase activity in parametrial adipose tissue, 12 units/g in control rats, increased 2-fold between the 9th and 12th days of pregnancy and then decreased slightly during the next 7 days, (1 unit of lipoprotein lipase activity = 1 μmole of triglyceride hydrolyzed per h.) It decreased on the 20th day to 5 units/g, reached 2 units/g at parturition, and remained below 2 units/g throughout lactation. 4. 4. Lipoprotein lipase activity in the inguinal-abdominal mammary glands increased very slowly during the first 20 days of pregnancy, from 0.9 units/g to 7 units/g. It then increased the next day to 21 units/g, decreased sharply at parturition to 6 units/g, and increased after parturition to more than 40 units/g. Lipoprotein lipase activity in mammary gland remained high as long as the mother suckled. 5. 5. Nonsuckling for 9 or more h decreased mammary gland lipoprotein lipase activity to near zero and increased plasma triglyceride concentration to 3 mM. Nonsuckling also increased lipoprotein lipase activity in adipose tissue. 6. 6. Ligation of the lactiferous ducts of the inguinal-abdominal glands on one side markedly reduced the lipoprotein lipase activity in these glands but not that in the contralateral, suckled glands. 7. 7. Plasma triglyceride concentration was inversely related to the lipoprotein lipase activity in mammary tissue during the last 2 days of pregnancy and throughout lactation. 8. 8. It is suggested that the changes in lipoprotein lipase activity of adipose and mammary tissues that occur during late pregnancy and lactation serve to divert dietary lipid from storage in adipose tissue to mammary glands for milk formation.

Effect of insulin and acute diabetes on plasma FFA and ketone bodies in the fasting rat

The metabolism of FFA and ketone bodies was studied in fasted rats by infusing at a constant rate tracer amounts of FFA-(3)H, beta-hydroxybutyrate-(14)C or acetoacetate-(14)C for periods up to 2 hr. Blood that was removed for analyses was replaced by continuous transfusion. The rates of turnover of FFA, beta-hydroxybutyrate, and acetoacetate in rats fasted for 2 days were, respectively, 3.2, 5.6, and 2.5 mumoles/100 g body weight per min. Infusion of mannoheptulose with anti-insulin serum increased plasma glucose, FFA, and ketone body concentrations and decreased the specific activity of plasma FFA. Injection of insulin (20 mU i.v.) decreased almost simultaneously plasma glucose, FFA, and ketone body concentrations and increased the specific activity of FFA, but it did not affect the plasma concentration of FFA-(3)H. The findings indicate that insulin deprivation increased and insulin injection decreased the release of FFA from body tissues in fasting rats. The plasma FFA concentration in fasting rats was increased by infusing chylomicrons and heparin, but this had very little effect on either plasma ketone body or glucose concentrations. Insulin injection (20 mU i.v.) lowered the plasma ketone body concentration in these animals. Studies using beta-hydroxybutyrate-(14)C showed that insulin (50 mU i.v.) decreased ketogenesis in the presence of a sustained high plasma FFA concentration and had no effect on uptake of circulating ketone bodies. The results indicate that plasma FFA concentration is not the sole determinant of plasma ketone body concentration and that insulin can suppress ketone body production through some means other than lowering plasma FFA concentration.

Effects of Mannoheptulose on Glucose Metabolism of Isolated Tissues

Summary The mechanism of the diabetogenic action of mannoheptulose has been investigated by testing its action on glucose metabolism by isolated tissues. Mannoheptulose did not alter the in vitro metabolism of glucose by liver, kidney, diaphragm or epididymal fat pad. The stimulating effects of insulin on glucose metabolism of diaphragm and adipose tissue were not decreased by mannoheptulose.

Lipoprotein lipase and uptake of triacylglycerol, cholesterol and phosphatidylcholine from chylomicrons by mammary and adipose tissue of lactating rats in vivo.

The relationship between lipoprotein lipase activity and uptake of triacylglycerol, cholesterol and phosphatidylcholine from chylomicrons was studied in mammary gland and adipose tissue of rats lactating 6--7 days. 60% of triacylglycerol [14C]oleic acid, 13% of [3H]cholesterol and 8% of [32P]phosphatidylcholine in chylomicrons injected intravenously were taken up within 11 min by mammary gland whereas negligible amounts were taken up by adipose tissue. Non-suckling for 44 h decreased markedly uptake of all lipids by mammary gland and retarded clearance of chylomicrons from blood, while it increased significantly uptake of triacylglycerol fatty acids and cholesterol by adipose tissue. Non-suckling also decreased lipoprotein lipase activity in mammary gland from 7.7 to 0.4 units/g, while it increased activity in adipose tissue from 0.1 to 2.7 units/g. These findings indicate that lipoprotein lipase is involved in uptake of chylomicron triacyglycerol and cholesterol by mammary gland and adipose tissue, and also in uptake of chylomicron phosphatidylcholine by mammary gland. They also show that reciprocal changes in lipoprotein lipase activity in mammary gland and adipose tissue, as occur during lactation, result in diversion of chylomicron lipids from one tissue to the other.

Ketosis in the rat fetus.

Summary Blood levels of ketone bodies, glucose, and fats in pregnant rats were altered by fasting and by pancreatectomy. The concentration of ketones and glucose in fetal blood changed with maternal level, whereas the lipids were independent of maternal level. Ketone bodies appear to cross the placenta as rapidly as glucose.

Early effects of ‘total’ pancreatectomy on fat metabolism in the rat

Within 2 hours after ‘total’ pancreatectomy in fasting rats blood concentrations of glucose, ketone bodies and total lipids were significantly elevated. Blood ketone bodies and total lipids increased before the blood glucose reached diabetic levels. The increase in blood lipids was due largely to triglycerides and was accompanied by accumulation of fat in the liver and kidneys. When depleted of body fat prior to surgery, pancreatectomized rats did not develop ketosis, hyperlipemia or fatty liver and kidneys. Repeated injections of insulin started immediately after pancreatectomy prevented development of diabetes. When insulin was given for the first time 1 day after pancreatectomy, blood glucose, ketone bodies and fats were lowered to normal fasting levels within 3 hours; 2 days after pancreatectomy, blood glucose and fats were reduced to normal levels whereas the ketonemia, although somewhat decreased, remained high. The rapid changes in blood ketone bodies are interpreted as evidence of a direct insulin action on liver. The decreased insulin effect on ketonemia in rats deprived of insulin for 2 days may indicate development of hepatic resistance to insulin.

Effect of Insulin on FFA Mobilization and Ketosis in Fasting Pregnant Rats

The concentrations of FFA and ketone bodies in plasma increased more in rats pregnant for eighteen days than in nonpregnant rats when both groups were fasted for two days. The immunoreactive insulin content of plasma was the same in fasted nonpregnant and pregnant rats whether the latter were ketotic or not. Hence, decreased circulating insulin, per se, did not appear to be the primary cause of ketosis in fasting pregnant rats. FFA mobilization was not suppressed completely by insulin in ketotic pregnant rats. The adipose tissue of pregnant rats showed impaired metabolism of glucose and increased FFA release in vitro. Lipolysis, in the presence or absence of insulin, was greater in adipose tissue of pregnant than in that of nonpregnant rats. Ketogenesis by liver slices of pregnant rats was greater than that of nonpregnant control rats and was not reduced thirty minutes after insulin injection despite a 50 per cent lowering of the concentration of plasma FFA and ketone bodies during this time. It is suggested that the ketosis of fasting in pregnant rats is caused by increased lipolysis in their adipose tissues and that increased FFA release is due to the absence of sufficient glucose to support re-esterification in the adipose tissues. The small effect of insulin (0.2 and 1.0 U., subcutaneously) on the plasma FFA may be due to the very low plasma glucose in fasting pregnant rats.

Effect of combined lipase deficiency (cld/cld) on hepatic and lipoprotein lipase activities in liver and plasma of newborn mice

Combined lipase deficiency (cld/cld) is a recessive mutation in mice which results in massive hyperlipemia and death within 3 days after birth. We studied the effect of this deficiency on lipolytic activities in liver and in pre- and postheparin plasma of mice less than 2 days old. Anti-hepatic lipase serum inhibited more than 85% of the lipolytic activity in liver and plasma of normal newborn mice when assayed in high-salt medium, validating the use of this medium for measuring hepatic lipase activity in mice. Anti-lipoprotein lipase serum, in contrast, inhibited only two-thirds of the lipolytic activity in liver and plasma when assayed in serum low-salt medium, and anti-hepatic lipase serum inhibited the rest. This indicates that assay with serum low-salt medium alone is not specific for lipoprotein lipase activity in mice. Therefore, immunoinhibition was used, as needed, for measuring lipoprotein lipase activity. The livers of unaffected newborn mice contained high levels of both hepatic and lipoprotein lipase activities, 228 and 187 mU/g, respectively. The plasma of unaffected mice contained a high level of hepatic lipase activity, 244 mU/ml, but practically no lipoprotein lipase activity. Heparin injected intraperitoneally increased plasma lipoprotein lipase activity to 152 mU/ml, but had no effect on plasma hepatic lipase activity, in unaffected mice. Hepatic lipase activity was virtually absent from both liver and plasma of cld/cld mice. Lipoprotein lipase activity was present in the liver at a surprisingly high level, 40% of that in normals, but was barely detectable in plasma. Heparin injection increased plasma lipoprotein lipase activity in cld/cld mice, but the increment was less than 10% of that in unaffected mice. Heparin had no significant effect on plasma hepatic lipase activity in defective mice. These findings confirm preliminary observations that hepatic lipase activity in liver and plasma and lipoprotein lipase activity in plasma are markedly reduced in combined lipase deficiency. The unexpected high level of lipoprotein lipase activity in liver of cld/cld mice suggests that regulation of lipoprotein lipase activity in liver of neonatal mice is different from that in other tissues.

Role of Lipolytic and Glucocorticoid Hormones in the Development of Diabetic Ketosis

Pancreatectomized rats hypophysectomized for one week or longer developed ketosis when given dexamethasone and growth hormone in amounts that were relatively inactive when administered alone. Growth hormone could be replaced by other lipolytic hormones, such as adrenocortico-trophic hormone (ACTH) and thyroxine. When fasted hypophysectomized rats were made acutely diabetic by injection of mannoheptulose, the plasma concentrations of glucose, ketone bodies, free fatty acids (FFA) and glycerol were increased; injection of growth hormone and dexamethasone further increased the concentrations of these plasma constituents. Injection of these hormones increased lipolysis and decreased glucose metabolism, in vitro, by adipose tissue of mannoheptulose-treated hypophysectomized rats. Uptake of FFA and ketogenesis by perfused livers of fasted hypophysectomized rats were directly related to the concentration of FFA in the perfusing fluid. Growth hormone and glucocorticoid did not have an effect on FFA uptake and ketogenesis in the liver. It is concluded that lipolytic and glucocorticoid hormones induce diabetic ketosis through their actions on adipose tissue. Lipolytic hormones stimulate lipolysis in adipose tissue while glucocorticoid limits, in the absence of insulin, re-esterification of FFA by suppressing the metabolism of glucose. The resultant increased release of FFA to the blood augments, in turn, the concentration of FFA in plasma, the uptake of FFA by the liver, and the formation of ketone bodies in the liver.

Effect of pH on visualization of fatty acids as myelin figures in mouse adipose tissue by freeze-fracture electron microscopy.

We studied the effect of pH on visualization of fatty acids as myelin figures in young mouse epididymal adipose tissue. Fatty acid content of the tissue was increased to 12.4 nmol/mg wet weight by treating the tissue with 380 microM isoproterenol at pH 7.4 for 15 min in the absence of glucose and albumin. Myelin figures were found in freeze-fracture replicas of isoproterenol-treated tissue fixed with glutaraldehyde at pH 7.4 and then incubated and glycerinated at pH 8.1. Myelin figures were seen in replicas as concave or convex laminated sheets and long cylindrical multilamellar structures in fat cells and extracellular space. Myelin figures were sometimes seen in cells extending from the surface of intracellular lipid droplets, the site of lipolysis, to the cell surface and extracellular space. Myelin figures were not found in isoproterenol-treated tissue fixed at pH 7.4 and processed at pH 7.0. Smooth-surfaced droplets, instead, were found in these tissues in the extracellular space. Neither myelin figures nor smooth-surfaced droplets were found in tissues treated with insulin and glucose (to reduce fatty acid content to 1.4 nmol/mg), fixed at pH 7.4 and processed at either pH 8.1 or pH 7.0. Lowering pH of the media to 4.5 during processing of tissues treated with isoproterenol at pH 9.0 caused disappearance of myelin figures and appearance of smooth-surfaced droplets in the extracellular space. Myelin figures were found in replicas of tissue treated with isoproterenol for 15 min at pH 7.4, incubated 10 min at pH 8.4, quick-frozen and then freeze-fractured, indicating that formation of myelin figures was not dependent on glutaraldehyde fixation and glycerol infiltration of the tissue. Our findings show that excess fatty acids in adipose tissue can be visualized as myelin figures if the tissue is exposed to pH 8.1-9.0 and maintained at or above pH 7.4, or as smooth-surfaced droplets if the tissue is processed at pH 7.0 or 4.5. We conclude that myelin figures formed under these conditions are composed primarily of partially ionized fatty acids (acid-soaps), and that the smooth-surfaced droplets in the extracellular space are composed of un-ionized (protonated) fatty acids.