Gerda Endemann

The oxidation of low density lipoprotein (LDL) in the arterial wall is thought to contribute to human atherosclerotic lesion formation, in part by the high affinity uptake of oxidized LDL (OxLDL) by macrophages, resulting in foam cell formation. We have utilized cloning by expression to identify CD36 as a macrophage receptor for OxLDL. Transfection of a CD36 clone into 293 cells results in the specific and high affinity binding of OxLDL, followed by its internalization and degradation. An anti-CD36 antibody blocks 50% of the binding of OxLDL to platelets and to human macrophage-like THP cells. Furthermore, like mouse macrophages, 293 cells expressing CD36 recognize LDL which has been oxidized only 4 h, whereas more extensive oxidation of the LDL is required for recognition by the other known OxLDL receptors, the acetylated LDL (AcLDL) receptor and Fc gamma RII-B2. CD36 may play a role in scavenging LDL modified by oxidation and may mediate effects of OxLDL on monocytes and platelets in atherosclerotic lesions.

The tyrosine kinase activity intrinsic to the insulin receptor is thought to be important in eliciting the intracellular responses to insulin; however, it has been difficult to determine the biochemical functions of the proteins which are substrates for this receptor. Treatment of Chinese hamster ovary (CHO) cells overexpressing the human insulin receptor (CHO.T) with insulin results in a 38 +/- 11 (mean +/- S.E., n = 9)-fold increase in a phosphatidylinositol (PtdIns) kinase activity in anti-phosphotyrosine immunoprecipitates of whole cell lysates. One minute of treatment of cells with insulin causes a dramatic increase in the PtdIns kinase activity in the anti-phosphotyrosine immunoprecipitates; the activity peaks within 5 min and remains elevated for at least 60 min after addition of insulin to the cells. This response is only slightly delayed compared with the time course we observe for activation of the insulin receptor tyrosine kinase. The insulin dose-response curves are also very similar for the activation of the insulin receptor tyrosine kinase activity and for the appearance of PtdIns kinase in the anti-phosphotyrosine immunoprecipitates. Stimulation of the endogenous insulin receptor of CHO cells also results in the association of PtdIns kinase activity with phosphotyrosine-containing proteins. However, CHO cells are less sensitive to insulin than CHO.T cells, and the maximal PtdIns kinase activity in antiphosphotyrosine immunoprecipitates from CHO cells is one-sixth that of CHO.T cells. In contrast, immunoprecipitates from CHO.T cells made with anti-insulin receptor antibodies do not contain significant levels of PtdIns kinase activity. This demonstrates that the PtdIns kinase is either a substrate for the insulin receptor tyrosine kinase or is tightly associated with another tyrosine phosphoprotein, which is not the insulin receptor.

The internalization of oxidized low density lipoprotein (OxLDL) by macrophages is hypothesized to contribute to foam cell formation and eventually to atherosclerotic lesion formation. OxLDL is a ligand for the acetylated low density lipoprotein (AcLDL) receptor, however, our data show that this receptor accounts for less than half of OxLDL uptake by mouse macrophages, suggesting additional receptors for OxLDL. We have developed a novel expression cloning strategy in order to isolate clones encoding OxLDL receptors. In addition to the AcLDL receptor, we isolated a molecular clone for a structurally unrelated receptor capable of mediating the high affinity uptake of OxLDL following transfection into cells. This receptor has been identified as the mouse Fc gamma RII-B2, a member of a family of receptors known to mediate immune complex uptake through recognition of the Fc region of IgG. The uptake of OxLDL by cells transfected with the Fc gamma RII-B2 clone is not blocked by AcLDL but is blocked by the anti-Fc gamma RII monoclonal antibody, 2.4G2.

The production of ketone bodies by the isolated perfused rat liver has been measured by the dilution of the specific activity of tracer amounts of beta-hydroxy[3-14C]butyrate and by accumulation in the perfusate. The latter method has been found to underestimate ketogenesis by 12 to 44% because it does not take into account acetoacetate utilization by the liver. Incorporation of ketone bodies into fatty acids and 3-beta-hydroxysterols was compared to total lipid synthesis measured by incorporation of tritium from tritiated water. A preferential labeling of 3-beta-hydroxysterols over fatty acids was observed, which is consistent with the activation of acetoacetate in the cytosol by acetoacetyl-CoA synthetase. Ketone bodies contribute 19 to 80% of the carbon incorporated into sterols and up to 22% of the carbon incorporated into fatty acids, depending upon the metabolic status of the liver. The activity of acetoacetyl-CoA synthetase is more than sufficient to account for the rate of ketone body utilization. Conditions that decrease the citrate cleavage pathway of acetyl group translocation through the mitochondrial membrane are associated with an increase in carbon flux through acetoacetyl-CoA synthetase. Formation of acetoacetate in the mitochondria and its utilization in the cytosol thus appear to be a secondary pathway of acetyl group translocation operating concurrently with the predominant citrate cleavage pathway.

Two phosphatidylinositol (PI) kinases from bovine brain were separated by rate zonal sucrose gradient centrifugation of detergent-solubilized membranes. Of the total PI kinase activity, 43% migrates on sucrose gradients with a size of approximately 55 kilodaltons (kDa); this kinase has properties similar to one of two PI kinase activities characterized in fibroblasts [Whitman, M., Kaplan, D. R., Roberts, T., & Cantley, L. (1987) Biochem. J. (in press)] and has been termed type 2. The remainder of the activity migrates in a second peak with a size of approximately 230 kDa. This enzyme possesses properties which are unlike both fibroblast PI kinase activities and has been termed type 3. The type 2 and type 3 enzymes have very different affinities for adenine nucleotides and are readily distinguishable by their sensitivities to inhibition by adenosine. The KMs of types 2 and 3 kinases for ATP are 54 and 742 microM, and the Kis for adenosine are 18 and 1520 microM, respectively. The two enzymes also differ in their affinities for PI, phosphatidylinositol 4-phosphate, and Mg2+ as well as in stimulation and inhibition by other phospholipids. When PI kinase from erythrocyte ghosts is fractionated by sucrose gradient centrifugation, only one peak of activity is observed which is indistinguishable from brain type 2 PI kinase.