Ray C. Pittman

Cholesterol esters selectively taken up from high-density lipoproteins are hydrolyzed extralysosomally

High-density lipoprotein (HDL) cholesterol esters (CE) are taken up by many cells without parallel uptake of HDL apoproteins. This selective uptake is mediated by reversible incorporation of HDL CE into a plasma membrane pool, from which the CE are internalized. We now show that selectively taken up CE are directed to an extralysosomal destination where they are hydrolyzed and available to the steroidogenic pathway. Cultured human fibroblasts take up HDL CE predominantly by selective uptake. Wolmans disease fibroblasts, which are deficient in lysosomal cholesterol esterase, effectively hydrolyzed CE from HDL, but not CE taken up in low density lipoproteins (LDL); normal fibroblasts hydrolyzed both effectively. Analogously, the lysosomotropic agent chloroquine effectively blocked hydrolysis of LDL CE but not HDL CE. A similar effect of chloroquine was seen in primary cultures of rat adrenal cells, which are very active in selective uptake. More than 50% of HDL CE taken up by adrenal cells appeared in the medium as corticosterone. To examine the subcellular destination of selectively taken up CE, non-hydrolyzable tracers of HDL and LDL CE were simultaneously injected into rats. On fractionation of adrenal glands 24 h after injection, 83% of the HDL CE tracer and 48% of the LDL CE tracer were recovered in cytoplasmic lipid droplets; that LDL tracer in the lipid droplets was accounted for by selective uptake of CE from LDL. Thus, selectively taken up cholesterol esters are processed by a mechanism distinct from the classical endosomal/lysosomal pathway, and are delivered to a cytoplasmic compartment.

A plasma membrane pool of cholesteryl esters that may mediate the selective uptake of cholesteryl esters from high-density lipoproteins.

Selective uptake of high-density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles occurs by a nonendocytotic pathway that requires no specific apolipoprotein and results in the net delivery of cholesteryl esters to cells. Here we examine a reversibly cell-associated pool of cholesteryl ester tracer and its relationship to selective uptake. A fraction of cholesteryl ester tracer selectively taken up from HDL by rat primary or mouse Y1-BS1 adrenocortical cells was chased from the cells by subsequent incubation with unlabeled HDL. This pool of cholesteryl ester tracer was distinct from that irreversibly internalized, and in excess of that accounted for by dissociation of labeled HDL particles bound to the cell surface. In response to various metabolic effectors, cholesteryl ester tracer in this reversibly cell-associated pool of Y1-BS1 cells correlated linearly with irreversible selective uptake. Both reversibly and irreversibly cell-associated pools of cholesteryl ester tracer displayed similar saturation kinetics for uptake from HDL, and both pools correlated inversely with cell-free cholesterol levels. Cholesteryl ester tracer in the reversible pool was shown to serve as a precursor for irreversible selective uptake. A pool with properties similar to the reversibly cell-associated pool was identified in plasma membrane fractions; enough tracer was incorporated into this pool to account for the reversibly cell-associated pool of intact cells. The data suggest that a pool of cholesteryl esters in the plasma membrane is involved in selective uptake at a step prior to irreversible internalization.

Tissue sites of catabolism of rat and human low density lipoproteins in rats

We have determined the sites of degradation of low density lipoprotein in rats using covalently linked [14C] sucrose as tracer. On degradation, 14C is trapped intracellularly as a cumulative measure of the amount of protein catabolized by each tissue. [14C] Sucrose-labeled rat low density lipoprotein (d 1.02-1.05 g/ml) was cleared from the plasma at a rate (0.092 +/- 0.003 h-1) similar to that for 125I-labeled LDL (0.096 +/- 0.22 h-1). Tissues were examined for total 14C content 24 h after injection of 14C-labeled lipoprotein. At death, animals were perfused thoroughly to remove trapped plasma. Recovery of 14C in tissue was 100 +/- 23% of catabolized 14C-labeled lipoprotein (calculated from plasma decay kinetics). In three test tissues, leakage of 14C over 5 days was less than 10%/day; leakage from liver was 10%/day, predominantly into bile; 14C content of kidney increased slightly. Thus, 14C trapping was adequate. The 14C-labeled lipoprotein was catabolized 66.8 +/- 2.5% by liver. No other organ catabolized more than 8%. Liver, adrenal and ovary were the most active per unit wet weight, followed by spleen. Urinary excretion, in 24h, was 3% and biliary excretion was 7% of catabolized. Human low density lipoproteins were similarly examined with similar results; this similarity may be due to exchange of rat apolipoproteins onto human lipoprotein in the circulation.

Partial purification and characterization of hormonesensitive lipase from rat adipose tissue

Abstract 1. 1. A high yield of triglyceride lipase was recovered reproducibly in the clear 105000 × g supernatant (S 105 fraction) when rat epididymal fat pads were homogenized in 0.25 M sucrose containing 10 −3 M EDTA. 2. 2. Previous exposure of intact fat pads to epinephrine (5 · 10 −5 M) caused an increment in the triglyceride lipase activity recovered in the S 105 fraction. 3. 3. This hormone-sensitive lipase activity was not inhibited by 1 M NaCl or protamine sulfate (800 μg/ml). NaF (10 −2 M) inhibited the activity by 80%. 4. 4. A part of the lipase activity floated during preparative ultracentrifugation at medium densities greater than 1.125. Previous treatment of the intact fat pads with epinephrine increased the activity in this floating ( d 5. 5. Lipase activity in the S 105 fraction was rapidly inactivated during incubation at 37° and pH 7.4. This deactivation could be blocked by 10 −3 M EDTA. 6. 6. After 35-fold purification of lipase, obtained by precipitation at pH 5.2 and preparative ultracentrifugation at d = 1.125, the enzyme was stable even in the absence of EDTA. 7. 7. A high level of lipoprotein lipase activity was demonstrable in the S 105 fraction when fat pads from fed rats were used. Fasting of the animals or preincubation of fat pads in glucose-free medium lowered this activity without changing the activity of the hormone-sensitive triglyceride lipase.

Activatable cholesterol esterase and triacylglycerol lipase activities of rat adrenal and their relationship

Activatable cholesterol esterase and triacylglycerol lipase of rat adrenal were 58-69% recovered in the 100 000 X g supernatant fraction. Activatable triacylglycerol lipase activity was differentiated from the activity of acid lipase and lipoprotein lipase also found in this fraction. Cholesterol esterase was activated 39.7 +/- 13.6% (S.D.) and triacylglycerol lipase 11.9 +/- 2.9% in a reaction dependent on ATP, cyclic AMP, and protein kinase. The two activities were shown by differential inhibition by an organophosphate, and by partial separation on salting out, to be largely due to separate enzymes. The two enzymes bound tightly to substrate emulsions with quantitatively similar distribution between competing emulsions, suggesting concerted binding. Coinciding gel filtration patterns reinforced, The hypothesis of a lipase complex. Cholesterol esterase comprised a major component of higher apparent Km for substrate and molecular weight 3-10(5)-6-10(5) by gel filtration and a minor component of lower apparent Km and heterogeneous molecular weight above 1 million, which was found mostly in complex and lipid.

A new approach for assessing cumulative lysosomal degradation of proteins or other macromolecules

Abstract A general method is proposed for the direct estimation of the degradation in various tissues of macromolecules that are metabolized by a lysosomal mechanism. This involves coupling to the macromolecule a small molecule that is cleaved from it only after entry into the lysosome, that is not metabolized but is “trapped” in the lysosome, and that therefore accumulates as a direct function of the amount of macromolecule degraded. The feasibility of the method was shown using low density lipoprotein and serum albumin doubly labeled with covalently bound [14C]sucrose and 125I. Uptake by normal fibroblasts, measured in terms of 14C accumulated in the cells, correlated very closely with uptake measured in terms of 125I-labeled metabolites in the medium plus 125I in the cells.

Acid acylhydrolase deficiency in I-cell disease and pseudo-Hurler polydystrophy.

Abstract Fibroblasts from patients with I-cell disease and pseudo-Hurler poly-dystrophy are characterized by multiple lysosomal enzyme deficiencies; only acid β- d -galactosidase and α- d -neuraminidase deficiency have been described in liver. Acid cholesterol ester hydrolase and acid triacylglycerol hydrolase have not previously been studied in either tissue. Acid cholesterol ester hydrolase activity in five lines of I-cell fibroblasts was 18 ± 5% (S.D.) of normal. Acid triacylglycerol hydrolase was 17 ± 3% of normal. Equivalence in deficiency of the two activities was also seen in fibro-blasts from patients with pseudo-Hurler poly dystrophy, Wolmans disease and cholesterol ester storage disease, suggesting that both activities reside in one enzyme. Although the apparent Km of cholesterol ester hydrolase (substrate emulsified with Triton X-100) was moderately elevated in two of three I-cell lines examined, deficiency was more a function of diminished catalytic capacity in all cases. In autopsied liver from three I-cell patients acid cholesterol ester hydrolase was 1–11% and acid triacylglycerol was 8–22% of normal; triacylglycerol hydrolase was less deficient in each case. In the liver of another I-cell patient, KZ, cholesterol ester hydrolase was 25–60% and triacylglycerol hydrolase was 86–194% of normal. Both activities in KZ fibroblasts were 17–20% of normal, indicating that the deficiency in fibroblasts does not necessarily denote triacylglycerol deficiency in liver. The apparent Km of liver cholesterol ester hydrolase was moderately elevated in two of the four samples.

A second form of hormone-sensitive lipase from rat adipose tissue homogenates.

Abstract Previous studies in this laboratory demonstrated the presence of a lipid-rich, hich molecular weight ( S 20, w = 34) form of hormone-sensitive lipase in rat adipose tissue. This lipase was purified about 100-fold from the 78000 × g supernatant fraction and shown to be activated by cyclic AMP-dependent protein kinase. The present studies demonstrate the presence in rat adipose tissue of a second form of hormonesensitive lipase, also protein kinase activatable, that is retained on 4% agarose columns, emerging with proteins of molecular weight of approx. 100000–200000. This retained lipase does not have the low density characteristic of the high molecular weight, lipidrich enzyme. Its relationship to the previously described lipase is under investigation.