Yuko Kako

Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance

Insulin resistance in skeletal muscle and liver may play a primary role in the development of type 2 diabetes mellitus, and the mechanism by which insulin resistance occurs may be related to alterations in fat metabolism. Transgenic mice with muscle- and liver-specific overexpression of lipoprotein lipase were studied during a 2-h hyperinsulinemic–euglycemic clamp to determine the effect of tissue-specific increase in fat on insulin action and signaling. Muscle–lipoprotein lipase mice had a 3-fold increase in muscle triglyceride content and were insulin resistant because of decreases in insulin-stimulated glucose uptake in skeletal muscle and insulin activation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity. In contrast, liver–lipoprotein lipase mice had a 2-fold increase in liver triglyceride content and were insulin resistant because of impaired ability of insulin to suppress endogenous glucose production associated with defects in insulin activation of insulin receptor substrate-2-associated phosphatidylinositol 3-kinase activity. These defects in insulin action and signaling were associated with increases in intracellular fatty acid-derived metabolites (i.e., diacylglycerol, fatty acyl CoA, ceramides). Our findings suggest a direct and causative relationship between the accumulation of intracellular fatty acid-derived metabolites and insulin resistance mediated via alterations in the insulin signaling pathway, independent of circulating adipocyte-derived hormones.

Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy

Lipoprotein lipase is the principal enzyme that hydrolyzes circulating triglycerides and liberates free fatty acids that can be used as energy by cardiac muscle. Although lipoprotein lipase is expressed by and is found on the surface of cardiomyocytes, its transfer to the luminal surface of endothelial cells is thought to be required for lipoprotein lipase actions. To study whether nontransferable lipoprotein lipase has physiological actions, we placed an alpha-myosin heavy-chain promoter upstream of a human lipoprotein lipase minigene construct with a glycosylphosphatidylinositol anchoring sequence on the carboxyl terminal region. Hearts of transgenic mice expressed the altered lipoprotein lipase, and the protein localized to the surface of cardiomyocytes. Hearts, but not postheparin plasma, of these mice contained human lipoprotein lipase activity. More lipid accumulated in hearts expressing the transgene; the myocytes were enlarged and exhibited abnormal architecture. Hearts of transgenic mice were dilated, and left ventricular systolic function was impaired. Thus, lipoprotein lipase expressed on the surface of cardiomyocytes can increase lipid uptake and produce cardiomyopathy.

Delayed catabolism of apoB-48 lipoproteins due to decreased heparan sulfate proteoglycan production in diabetic mice

We used wild-type (WT) mice and mice engineered to express either apoB-100 only (B100 mice) or apoB-48 only (B48 mice) to examine the effects of streptozotocin-induced diabetes (DM) on apoB-100- and apoB-48-containing lipoproteins. Plasma lipids increased with DM in WT mice, and fat tolerance was markedly impaired. Lipoprotein profiles showed increased levels and cholesterol enrichment of VLDL in diabetic B48 mice but not in B100 mice. C apolipoproteins, in particular apoC-I in VLDL, were increased. To investigate the basis of the increase in apoB-48 lipoproteins in streptozotocin-treated animals, we characterized several parameters of lipoprotein metabolism. Triglyceride and apoB production rates were normal, as were plasma lipase activity, VLDL glycosaminoglycan binding, and VLDL lipolysis. However, beta-VLDL clearance decreased due to decreased trapping by the liver. Whereas LRP activity was normal, livers from treated mice incorporated significantly less sulfate into heparan sulfate proteoglycans (HSPG) than did controls. Hepatoma (HepG2) cells and endothelial cells cultured in high glucose also showed decreased sulfate and glucosamine incorporation into HSPG. Western blots of livers from diabetic mice showed a decrease in the HSPG core protein, perlecan. Delayed clearance of postprandial apoB-48-containing lipoproteins in DM appears to be due to decreased hepatic perlecan HSPG.

Lipoprotein lipase-mediated selective uptake from low density lipoprotein requires cell surface proteoglycans and is independent of scavenger receptor class B type 1.

Lipoprotein lipase (LpL) hydrolyzes chylomicron and very low density lipoprotein triglycerides to provide fatty acids to tissues. Aside from its lipolytic activity, LpL promotes lipoprotein uptake by increasing the association of these particles with cell surfaces allowing for the internalization by receptors and proteoglycans. Recent studies also indicate that LpL stimulates selective uptake of lipids from high density lipoprotein (HDL) and very low density lipoprotein. To study whether LpL can mediate selective uptake of lipids from low density lipoprotein (LDL), LpL was incubated with LDL receptor negative fibroblasts, and the uptake of LDL protein, labeled with 125I, and cholesteryl esters traced with [3H]cholesteryl oleoyl ether, was compared. LpL mediated greater uptake of [3H]cholesteryl oleoyl ether than 125I-LDL protein, a result that indicated selective lipid uptake. Lipid enrichment of cells was confirmed by measuring cellular cholesterol mass. LpL-mediated LDL selective uptake was not affected by the LpL inhibitor tetrahydrolipstatin but was nearly abolished by heparin, monoclonal anti-LpL antibodies, or chlorate treatment of cells and was not found using proteoglycan-deficient Chinese hamster ovary cells. Selective uptake from HDL, but not LDL, was 2–3-fold greater in scavenger receptor class B type I overexpressing cells (SR-BI cells) than compared control cells. LpL, however, induced similar increases in selective uptake from LDL and HDL in either control or SR-BI cells, indicative of the SR-BI-independent pathway. This was further supported by ability of LpL to promote selective uptake from LDL in human embryonal kidney 293 cells, cells that do not express SR-BI. In Chinese hamster ovary cell lines that overexpress LpL, we also found that selective uptake from LDL was induced by both endogenous and exogenous LpL. Transgenic mice that overexpress human LpL via a muscle creatine kinase promoter had more LDL selective uptake in muscle than did wild type mice. In summary LpL stimulates selective uptake of cholesteryl esters from LDL via pathways that are distinct from SR-BI. Moreover this process also occurs in vivo in tissues where abundant LpL is present.

Heparin-binding defective lipoprotein lipase is unstable and causes abnormalities in lipid delivery to tissues

Lipoprotein lipase (LpL) binding to heparan sulfate proteoglycans (HSPGs) is hypothesized to stabilize the enzyme, localize LpL in specific capillary beds, and route lipoprotein lipids to the underlying tissues. To test these hypotheses in vivo, we created mice expressing a human LpL minigene (hLpL(HBM)) carrying a mutated heparin-binding site. Three basic amino acids in the carboxyl terminal region of LpL were mutated, yielding an active enzyme with reduced heparin binding. Mice expressing hLpL(HBM) accumulated inactive human LpL (hLpL) protein in preheparin blood. hLpL(HBM) rapidly lost activity during a 37 degrees C incubation, confirming a requirement for heparin binding to stabilize LPL: Nevertheless, expression of hLpL(HBM) prevented the neonatal demise of LpL knockout mice. On the LpL-deficient background hLpL(HBM) expression led to defective targeting of lipids to tissues. Compared with mice expressing native hLpL in the muscle, hLpL(HBM) transgenic mice had increased postprandial FFAs, decreased lipid uptake in muscle tissue, and increased lipid uptake in kidneys. Thus, heparin association is required for LpL stability and normal physiologic functions. These experiments confirm in vivo that association with HSPGs can provide a means to maintain proteins in their stable conformations and to anchor them at sites where their activity is required.

The Heparin-Binding Proteins Apolipoprotein E and Lipoprotein Lipase Enhance Cellular Proteoglycan Production

Apolipoprotein E (apoE) and lipoprotein lipase (LPL), key proteins in the regulation of lipoprotein metabolism, bind with high affinity to heparin and cell-surface heparan sulfate proteoglycan (HSPG). In the present study, we tested whether the expression of apoE or LPL would modulate proteoglycan (PG) metabolism in cells. Two apoE-expressing cells, macrophages and fibroblasts, and LPL-expressing Chinese hamster ovary (CHO) cells were used to study the effect of apoE and LPL on PG production. Cellular PGs were metabolically labeled with (35)[S]sulfate for 20 hours, and medium, pericellular PGs, and intracellular PGs were assessed. In all transfected cells, PG levels in the 3 pools increased 1.6- to 3-fold when compared with control cells. Initial PG production was assessed from the time of addition of radiolabeled sulfate; at 1 hour, there was no difference in PG synthesis by apoE-expressing cells when compared with control cells. After 1 hour, apoE-expressing cells had significantly greater production of PGs. Total production assessed with [(3)H]glucosamine was also increased. This was due to an increase in the length of the glycosaminoglycan chains. To assess whether the increase in PGs was due to a decrease in PG degradation, a pulse-chase experiment was performed. Loss of sulfate-labeled pericellular PGs was similar in apoE and control cells, but more labeled PGs appeared in the medium of the apoE-expressing cells. Addition of exogenous apoE and anti-human apoE antibody to both non-apoE-expressing and apoE-expressing cells did not alter PG production. Moreover, LPL addition did not alter cell-surface PG metabolism. These results show that enhanced gene expression of apoE and LPL increases cellular PG production. We postulate that such changes in vascular PGs can affect the atherogenic potential of arteries.

Apolipoprotein E Containing High Density Lipoprotein Stimulates Endothelial Production of Heparan Sulfate Rich in Biologically Active Heparin-like Domains A POTENTIAL MECHANISM FOR THE ANTI-ATHEROGENIC ACTIONS OF VASCULAR APOLIPOPROTEIN E

Reduced heparin and heparan sulfate (HS) proteoglycans (PG) have been observed in both inflammation and atherosclerosis. Methods to increase endogenous heparin and heparan sulfate are not known. We found that incubation of endothelial cells with 500–1,000 μg/ml high density lipoprotein (HDL) increased35SO4 incorporation into PG by 1.5–2.5-fold. A major portion of this increase was in HS and was the result of increased synthesis. Total PG core proteins were not altered by HDL; however, the ratio of 35SO4 to [3H]glucosamine was increased by HDL, suggesting increased sulfation of glycosaminoglycans. In addition, HDL increased the amount of highly sulfated heparin-like HS in the subendothelial matrix. HS from HDL-treated cells bound 40 ± 5% more125I-antithrombin III (requires 3-O sulfated HS) and 49 ± 3% fewer monocytes. Moreover, the HS isolated from HDL-treated cells inhibited smooth muscle cell proliferation (by 83 ± 5%) better than control HS (56 ± 6%) and heparin (42 ± 6%). HDL isolated from apolipoprotein E (apoE)-null mice did not stimulate HS production unless apoE was added. ApoE also stimulated HS production in the absence of HDL. ApoE did not increase35SO4 incorporation in macrophages and fibroblasts, suggesting that this is an endothelial cell-specific process. Receptor-associated protein inhibited apoE-mediated stimulation of HS only at higher (20 μg/ml) doses, suggesting the involvement of a receptor-associated protein-sensitive pathway in mediating apoE actions. In summary, our data identify a novel mechanism by which apoE and apoE-containing HDL can be anti-atherogenic. Identification of specific apoE peptides that stimulate endothelial heparin/HS production may have important therapeutic applications.

Multiple pathways ensure retinoid delivery to milk: studies in genetically modified mice

Retinoids are absolutely required for normal growth and development during the postnatal period. We studied the delivery of retinoids to milk, availing of mouse models modified for proteins thought to be essential for this process. Milk retinyl esters were markedly altered in mice lacking the enzyme lecithin:retinol acyltransferase (Lrat(-/-)), indicating that this enzyme is normally responsible for the majority of retinyl esters incorporated into milk and not an acyl-CoA dependent enzyme, as proposed in the literature. Unlike wild-type milk, much of the retinoid in Lrat(-/-) milk is unesterified retinol, not retinyl ester. The composition of the residual retinyl ester present in Lrat(-/-) milk was altered from predominantly retinyl palmitate and stearate to retinyl oleate and medium chain retinyl esters. This was accompanied by increased palmitate and decreased oleate in Lrat(-/-) milk triglycerides. In other studies, we investigated the role of retinol-binding protein in retinoid delivery for milk formation. We found that Rbp(-/-) mice maintain milk retinoid concentrations similar to those in matched wild-type mice. This appears to arise due to greater postprandial delivery of retinoid, a lipoprotein lipase (LPL)-dependent pathway. Importantly, LPL also acts to assure delivery of long-chain fatty acids (LCFA) to milk. The fatty acid transporter CD36 also facilitated LCFA but not retinoid incorporation into milk. Our data show that compensatory pathways for the delivery of retinoids ensure their optimal delivery and that LRAT is the most important enzyme for milk retinyl ester formation.

A SELECTIVE INHIBITOR OF INTESTINAL ACAT, EAB309 SUPPRESSES BOTH INTESTINAL AND HEPATIC CHOLESTEROL OUTPUT AND STIMULATES CHYLOMICRON REMOVAL

The effect of a novel inhibitor of acylcoenzyme A:cholesterol acyltransferase (EC 2.3.1.26, ACAT), EAB309 (EAB) on plasma lipid metabolism was studied in cholesterol-fed rats. Orally administered EAB was not detected in the portal vein or the liver but distributed exclusively in the intestine, suggesting that this agent selectively inhibits intestinal ACAT. The rats were fed with either a cholesterol-diet or a cholesterol-diet containing 0.005% EAB (w/w) ad. libium for three weeks. ACAT activity in intestinal microsomes was significantly inhibited in EAB-treated rats. Hepatic ACAT activity was also decreased in EAB-treated rats, however, this was attenuated by the addition of excess cholesterol to the liver microsome, indicating that substrate availability is tightly associated with this enzymes activity and the inhibition of hepatic ACAT by EAB is not direct. Incorporation of [3H]-cholesterol to cholesteryl ester (CE) in mesenteric lymph were markedly suppressed by EAB treatment. Chylomicrons (CMs) were doubly labeled with [3H]-vitamin A and [14C]-triglyceride (TG) in EAB-treated or non-treated rats and injected into normal chow-fed rats. The CMs from EAB-treated rats were cleared faster from the plasma and taken up more by the liver compared with the CMs from non-treated rats. The content of CE in newly secreted VLDL was remarkably decreased by EAB treatment without affecting TG output. These results demonstrate that EAB, a novel inhibitor of intestinal ACAT, significantly suppresses both intestinal and hepatic CE output and stimulates CM removal. This suggests that the inhibition of intestinal ACAT can subsequently suppress hepatic ACAT by decreased CE delivery from the intestine to the liver.