Intracellular Lipoprotein Lipase Research Articles

Ghrelin Promotes Proliferation and Inhibits Differentiation of 3T3-L1 and Human Primary Preadipocytes.

ObjectiveGhrelin is a 28-amino-acid peptide that regulates energy hemostasis, glucose and lipid metabolism. We aimed to explore the effects of ghrelin on the proliferation and differentiation of 3T3-L1 and human primary preadipocytes.Methods3-(4,5-Dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) spectrophotometry, Oil Red O staining, intracellular glycerol-3-phosphate dehydrogenase (G-3-PDH) assays and semiquantitative reverse transcription polymerase chain reaction were used to investigate the action of ghrelin.ResultsGhrelin (0.01–1000 ng/ml) significantly increased the numbers of 3T3-L1 cells, and the maximum stimulatory effect was observed with the 10 ng/ml ghrelin treatment for 24 h (p < 0.05). Ghrelin also promoted the proliferation of human primary preadipocytes from 24 h (p < 0.05) to 48 h (p < 0.05) at a concentration of 1000 ng/ml. Further investigation showed that IGF-1 levels were notably increased in ghrelin-treated 3T3-L1 and human preadipocytes, and IGF-1 antibody was capable to attenuate this stimulatory action of ghrelin (all p < 0.05). Additionally, ghrelin significantly suppressed the differentiation of 3T3-L1 and human primary preadipocytes; 10 ng/ml ghrelin notably downregulated G-3-PDH activities in 3T3-L1 cells on day 3 and in human cells from days 4 to 12 following differentiation (all p < 0.05), and the intracellular lipoprotein lipase mRNA levels were lower than that of the controls (p < 0.05). Further investigation showed that the mRNA levels of peroxisome proliferator-activated receptor γ2 (PPARγ2) and CCAAT/enhancer binding protein α (C/EBPs) were also suppressed in ghrelin-treated human differentiating adipocytes.ConclusionGhrelin promotes the proliferation of 3T3-L1 and human primary preadipocytes by increasing the expression of IGF-1. Ghrelin inhibits murine and human adipocyte differentiation by downregulating PPARγ2 and C/EBPα levels, consequently leading to decreased lipid accumulation and lipogenic enzymes expression.

Suppression of skeletal muscle lipoprotein lipase activity during physical inactivity: a molecular reason to maintain daily low-intensity activity.

We have examined the regulation of lipoprotein lipase (LPL) activity in skeletal muscle during physical inactivity in comparison to low-intensity contractile activity of ambulatory controls. From studies acutely preventing ambulatory activity of one or both the hindlimbs in rats, it was shown that approximately 90-95 % of the heparin-releasable (HR) LPL activity normally present in rat muscle with ambulatory activity is lost, and thus dependent on local contractile activity. Similarly, approximately 95 % of the differences in LPL activity between muscles of different fibre types was dependent on ambulatory activity. The robustness of the finding that physical inactivity significantly decreases muscle LPL activity was evident from confirmatory studies with different models of inactivity, in many rats and mice, both sexes, three muscle types and during both acute and chronic (11 days) treatment. Inactivity caused a local reduction of plasma [3H]triglyceride uptake into muscle and a decrease in high density lipoprotein cholesterol concentration. LPL mRNA was not differentially expressed between ambulatory controls and either the acutely or chronically inactive groups. Instead, the process involved a rapid loss of the HR-LPL protein mass (the portion of LPL largely associated with the vascular endothelium) by an actinomycin D-sensitive signalling mechanism (i.e. transcriptionally dependent process). Significant decreases of intracellular LPL protein content lagged behind the loss of HR-LPL protein. Treadmill walking raised LPL activity approximately 8-fold (P < 0.01) within 4 h after inactivity. The striking sensitivity of muscle LPL to inactivity and low-intensity contractile activity may provide one piece of the puzzle for why inactivity is a risk factor for metabolic diseases and why even non-vigorous activity provides marked protection against disorders involving poor lipid metabolism.

Short-Term Intake of Conjugated Linoleic Acid Inhibits Lipoprotein Lipase and Glucose Metabolism but Does Not Enhance Lipolysis in Mouse Adipose Tissue1

Feeding diets supplemented with t10c12 conjugated linoleic acid (CLA) to growing mice reduces body fat mass. The effects are evident after 1 wk and maximal by 3 wk and are accompanied by reductions in fat cell size. This may complicate direct comparisons with adipocytes from control mice. Accordingly, we investigated the early biochemical events that occur within adipocytes during the first week of CLA feeding, before changes in the size of adipocytes have occurred. Female ICR mice were fed a control diet or a diet supplemented with 0.5 g/100 g of CLA for 4 d, at which time there were no differences in body weight, fat mass or adipocyte size (except that CLA-fed mice had fewer adipocytes >90 micro m in diameter). Parametrial adipose tissue from the CLA-fed mice had significantly reduced heparin-releasable lipoprotein lipase (LPL) and intracellular LPL activities and significantly reduced glucose incorporation into CO(2), fatty acid and glycerol. There were no differences between adipose tissues from CLA-fed or control mice in the ratios of 16:0 to 16:1 and 18:0 to 18:1 fatty acids or in norepinephrine-stimulated lipolysis. Serum insulin levels in food-deprived mice, measured at 4 d and 7 wk, did not differ between groups nor did the concentration of free fatty acids in serum of food-deprived or fed mice measured at the same time points. In mice, CLA-induced inhibition of heparin-releasable LPL and glucose metabolism may be the most important early steps leading to subsequent body fat reduction. In addition, CLA does not appear to enhance lipolysis in mouse adipose tissue in vivo.

Time course of changes in lipoprotein lipase activity in rat skeletal muscles during denervation-reinnervation.

The effects of denervation-reinnervation after sciatic nerve crush on the activity of extracellular and intracellular lipoprotein lipase (LPL) were examined in the soleus and red portion of gastrocnemius muscles. The activity of both LPL fractions was decreased in the two muscles within 24 h after the nerve crush and remained reduced for up to 2 wk. During the reinnervation period, LPL activity was still reduced in the soleus and started to increase only on the 40th day. In the red gastrocnemius, LPL activity increased progressively with reinnervation, exceeding control values on the 30th day post-crush. The LPL activity in the soleus from the contralateral to denervated hindlimb was also affected, being increased on the postoperation day and then gradually decreased during the following days. In conclusion, the time course of changes in muscle LPL activity after nerve crush confirmed the predominant role of nerve conduction in controlling muscle potential to take up free fatty acids derived from the plasma triacylglycerols. However, other factors, such as muscle fiber composition and the fiber transformation, should also be considered in this aspect of the denervation-reinnervation process. Moreover, it was found that denervation of muscles from one hindlimb may influence LPL activity in muscles from the contralateral leg.

Glucose and Insulin Stimulate Heparin-releasable Lipoprotein Lipase Activity in Mouse Islets and INS-1 Cells: A POTENTIAL LINK BETWEEN INSULIN RESISTANCE AND β-CELL DYSFUNCTION

Lipoprotein lipase (LpL) provides tissues with triglyceride-derived fatty acids. Fatty acids affect beta-cell function, and LpL overexpression decreases insulin secretion in cell lines, but whether LpL is regulated in beta-cells is unknown. To test the hypothesis that glucose and insulin regulate LpL activity in beta-cells, we studied pancreatic islets and INS-1 cells. Acute exposure of beta-cells to physiological concentrations of glucose stimulated both total cellular LpL activity and heparin-releasable LpL activity. Glucose had no effect on total LpL protein mass but instead promoted the appearance of LpL protein in a heparin-releasable fraction, suggesting that glucose stimulates the translocation of LpL from intracellular to extracellular sites in beta-cells. The induction of heparin-releasable LpL activity was unaffected by treatment with diazoxide, an inhibitor of insulin exocytosis that does not alter glucose metabolism but was blocked by conditions that inhibit glucose metabolism. In vitro hyperinsulinemia had no effect on LpL activity in the presence of low concentrations of glucose but increased LpL activity in the presence of 20 mm glucose. Using dual-laser confocal microscopy, we detected intracellular LpL in vesicles distinct from those containing insulin. LpL was also detected at the cell surface and was displaced from this site by heparin in dispersed islets and INS-1 cells. These results show that glucose metabolism controls the trafficking of LpL activity in beta-cells independent of insulin secretion. They suggest that hyperglycemia and hyperinsulinemia associated with insulin resistance may contribute to progressive beta-cell dysfunction by increasing LpL-mediated delivery of lipid to islets.

Direct regulatory effect of fatty acids on macrophage lipoprotein lipase: potential role of PPARs.

Atherosclerosis is a major complication of type 2 diabetes. The pathogenesis of this complication is poorly understood, but it clearly involves production in the vascular wall of macrophage (Mo) lipoprotein lipase (LPL). Mo LPL is increased in human diabetes. Peripheral factors dysregulated in diabetes, including glucose and free fatty acids (FAs), may contribute to this alteration. We previously reported that high glucose stimulates LPL production in both J774 murine and human Mo. In the present study, we evaluated the direct effect of FAs on murine Mo LPL expression and examined the involvement of peroxisome proliferator-activated receptors (PPARs) in this effect. J774 Mo were cultured for 24 h with 0.2 mmol/l unsaturated FAs (arachidonic [AA], eicosapentaenoic [EPA], and linoleic acids [LA]) and monounsaturated (oleic acid [OA]) and saturated FAs (palmitic acid [PA] and stearic acid [SA]) bound to 2% bovine serum albumin. At the end of this incubation period, Mo LPL mRNA expression, immunoreactive mass, activity, and synthetic rate were measured. Incubation of J774 cells with LA, PA, and SA significantly increased Mo LPL mRNA expression. In contrast, exposure of these cells to AA and EPA dramatically decreased this parameter. All FAs, with the exception of EPA and OA, increased extra- and intracellular LPL immunoreactive mass and activity. Intracellular LPL mass and activity paralleled extracellular LPL mass and activity in all FA-treated cells. In Mo exposed to AA, LA, and PA, an increase in Mo LPL synthetic rate was observed. To evaluate the role of PPARs in the modulatory effect of FAs on Mo LPL gene expression, DNA binding assays were performed. Results of these experiments demonstrate an enhanced binding of nuclear proteins extracted from all FA-treated Mo to the peroxisome proliferator-response element (PPRE) consensus sequence of the LPL promoter. PA-, SA-, and OA-stimulated binding activity was effectively diminished by immunoprecipitation of the nuclear proteins with anti-PPAR-alpha antibodies. In contrast, anti-PPAR-gamma antibodies only significantly decreased AA-induced binding activity. Overall, these results provide the first evidence for a direct regulatory effect of FAs on Mo LPL and suggest a potential role of PPARs in the regulation of Mo LPL gene expression by FAs.

Different effects of conjugated linoleic acid isomers on lipoprotein lipase activity in 3T3-L1 adipocytes

Conjugated linoleic acids (CLAs) are the positional and geometric isomers of linoleic acid. In the present study the effects of cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA ) on intracellular and heparin-releasable (HR-) lipoprotein lipase (LPL) activity in 3T3-L1 adipocytes were investigated. Cells were exposed to the two CLA isomers and linoleic acid, which were bound to bovine serum albumin (BSA). In the adipocytes insulin up-regulated and tumor necrosis factor alpha (TNFα) down-regulated HR-LPL activity, which corresponds with the findings in vivo. The experimental fatty acids at low concentrations (<30 μmol/L) moderately increased intracellular and HR-LPL activity. At a concentration of 100 μmol/L, c9,t11 CLA and t10,c12 CLA suppressed HR-LPL activity to 20 and 24% below the BSA control level, respectively, while linoleic acid had no effect unless its concentration was as high as 1000 μmol/L. Insulin abolished the inhibitory effect of c9,t11 CLA, but not of t10,c12 CLA. In the presence of insulin, t10,c12 CLA inhibited HR-LPL activity by 41% compared to BSA control. In contrast to TNFα, which suppressed both intracellular LPL and HR-LPL activity, CLAs suppressed HR-LPL activity without decreasing intracellular LPL activity. Additionally, t10,c12 CLA (100 μmol/L) partially prevented TNFα-induced decrease of intracellular LPL activity. These results indicate that CLAs differ from linoleic acid in regulating HR-LPL activity, and t10,c12 CLA appeared to be more effective than c9,t11 CLA.

Increased Intracellular Triglyceride in C2C12 Muscle Cells Transfected with Human Lipoprotein Lipase

Much of the knowledge about the cell biology of lipoprotein lipase (LPL) in vitro has been gained from adipose tissue model systems. However, the importance of skeletal muscle lipoprotein lipase (SMLPL) to both lipoprotein and muscle metabolism remains unclear. Although the production of LPL in cultured myocytes has been documented, the amount of enzyme activity produced is small. To develop a more suitable tissue culture model for SMLPL, mouse C2C12 myoblasts were stably transduced with a retroviral vector encoding the full-length human LPL (hLPL) cDNA. Control cells were transduced with a vector encoding β-galactosidase. LPL expression was assayed as a function of cell growth by measuring LPL activity on days 3, 7, 9, 11, and 14 after subculture. The hLPL-transduced myoblasts increasingly overexpressed both heparin-releasable (HR) and intracellular (IN) LPL activity compared to nontransduced myoblasts (P < 0.001 at Day 11) and myoblasts transduced with the control vector (P < 0.001 at Day 11). This increase occurred while LPL mRNA levels remained stable between days 3 and 14. As expected, IN LPL activity was also increased in the transduced cells. High levels of LPL activity were also obtained after differentiating the C2C12 cells into myotubes by serum deprivation. Additionally, throughout the time course, C2/LPL cells had greater amounts of intracellular triglyceride than both the C2C12 and the C2/β-GEO cells (P = 0.005 and P < 0.001, respectively) with the largest differences seen on day 14 of the time course (P = 0.001, C2/LPL vs C2C12r or C2/β-GEO cells). Thus, C2C12 myoblasts stably transduced with hLPL markedly overexpressed both HR and IN LPL activity compared to control cells which, in turn, was associated with increases in intracellular triglyceride content. Because LPL regulation in tissues is mostly posttranslational, this new in vitro model will permit the in-depth study of the posttranslational regulation of SMLPL and provide new insights into the fate of lipoprotein-derived fatty acids in muscle.

Binding and intracellular trafficking of lipoprotein lipase and triacylglycerol-rich lipoproteins by liver cells

The cellular mechanisms and pathways by which lipoprotein lipase (LPL) enhances the binding and uptake of lipoproteins remains unknown. Confocal and immunoelectron microscopy demonstrated that primary binding of bovine LPL (bLPL) occurs at the microvilli surface of HepG2 cells and hepatocytes. Internalized bLPL was associated with endocytic vesicles and multivesicular bodies. Quantitative immunofluorescence indicated that the presence of bLPL caused a marked increase in the cell-surface binding of DiI-conjugated triacylglycerol-rich lipoproteins (DiI-TRL). Confocal microscopy showed that when DiI-TRL was incubated with bLPL at 4°C, the distributions of bound LPL and DiI-TRL were totally coincident, and covered the apical surface of both HepG2 cells and hepatocytes. When incubated separately, the time-courses of the internalization of fluorescence associated with DiI-TRL and bLPL were different: DiI-TRL was quickly internalized by both HepG2 cells and hepatocytes, and reached a plateau at 30 min, whereas intracellular LPL increased continuously, but more slowly in the same period. In the presence of bLPL, DiI-TRL was internalized progressively by HepG2 and by cultured hepatocytes for up to 1 h and no saturation was reached. At this time the intensity of labeling of bLPL was lower than of DiI-TRL and a higher number of DiI spots did not colocalize with bLPL immunofluorescence, suggesting that the ligands follow a different pathway after internalization. The data suggest that when lipoprotein lipase (LPL) is associated with the lipoproteins it directs them to specific endocytic pathways. A hypothetical model of the intracellular pathways followed by triacylglycerol-rich lipoproteins and LPL after internalization is proposed.—Casaroli-Marano, R. P., R. García, E. Vilella, G. Olivecrona, M. Reina, and S. Vilaró. Binding and intracellular trafficking of lipoprotein lipase and triacylglycerol-rich lipoproteins by liver cells. J. Lipid Res. 1998. 39: 789–806.

Cardiac lipoprotein lipase in the spontaneously hypertensive rat.

The objectives of the present study were to determine functional (i.e., heparin-releasable) and intracellular (i.e., heparin-non-releasable) cardiac lipoprotein lipase (LPL) activity during the development of hypertension in spontaneously hypertensive (SHR) rats. Male WKY and SHR rats were killed before (7-8 weeks of age) and following (15-16 weeks of age) the development of severe hypertension in SHR rats. LPL activity in coronary perfusates was determined by retrogradely perfusing the hearts with heparin (5 U/ml). Cardiac myocytes were also isolated from the two groups of rats by collagenase digestion, and surface-bound and intracellular LPL activity measured. With the development of hypertension in SHR rats, there was a concomitant and progressive reduction in the heparin-releasable coronary endothelial LPL activity. Neither insulin action nor cell-associated enzyme activity could explain this low LPL activity in coronary blood vessels. However, acute vasodilation with nifedipine (a Ca2+ influx blocker) or CGS-21680 (A2-purinergic receptor agonist) increased the peak heparin-releasable LPL activity in hearts isolated from SHR rats. Our results indicate that hypertension per se may play a significant role in regulating cardiac LPL activity, and hence fatty acid supply to the hypertensive SHR rat heart.

Absence of N-glycosylation at asparagine 43 in human lipoprotein lipase induces its accumulation in the rough endoplasmic reticulum and alters this cellular compartment.

Lipoprotein lipase (LPL) is the enzyme responsible for the hydrolysis of plasma triglycerides from apolipoprotein C-II-containing lipoproteins at the capillary endothelium and it is synthesized in parenchymal cells of several tissues. Intracellular LPL processing is a major aspect of LPL regulation. The present study aims to determine the intracellular accumulation site of the LPL that is not glycosylated at Asn43. Human LPL (hLPL) cDNA was mutated by site-directed mutagenesis. An Ala residue was substituted for Asn at position 43 of the protein generating N43A hLPL. Wild type hLPL and the mutant hLPL were expressed in COS1 cells. Using immunofluorescence and immunoelectron microscopy we found that wild type hLPL in addition to being secreted into the medium was present in the rough endoplasmic reticulum (ER), Golgi compartments, and vesicles. Neither LPL activity nor protein was found in medium of cells expressing the mutant hLPL and all detectable protein was present exclusively in the ER identified witha specific antibody against the protein disulfide isomerase (PDI), an ER marker. In addition, the intracellular distribution of the ER of the cells that expressed the mutant protein was grossly altered. Treatment of COS1 cells with tunicamycin for 24 h had the same effect on wild type hLPL processing and edoplasmic reticulum distribution. Next, we investigated the influence of the accumulation of mutant hLPL on the intracellular transport of three other proteins that are N-glycosylated before reaching the plasma membrane: the related Bo,+ amino acid transporter (rBAT), the insulin-regulated glucose transporter (GLUT4), and the placental alkaline phosphatase (PLAP) protein. Coexpression of the mutant hLPL (but not wild type) caused the accumulation of rBAT and GLUT4 in the ER while PLAP reached the plasma membrane. Our findings demonstrate that glycosylation of Asn43 of human lipoprotein lipase in the endoplasmic reticulum is essential for its efflux from this compartment and that the retention of the non-glycosylated LPL induces morphological changes in the ER that could also affect its ability to modify the transport of other proteins.

Long-chain fatty acids decrease lipoprotein lipase activity of cultured rat adipocyte precursors

The effect of fatty acids on rat adipocyte precursor lipoprotein lipase (LPL) activity was examined. Cellular LPL activity in cultured perirenal precursors reached a maximum after 6 days. At day 6, addition of 10 −8 mol/L oleic acid to the culture medium for 6 hours resulted in a significant reduction of LPL activity. Exposing cultured precursors to 10 −4 mol/L oleic acid caused more than a 50% decrease of intracellular LPL activity measured in either acetone-ether or detergent extracts and more than a 60% decrease of heparin-releasable LPL activity. These reductions were evident within 2.5 hours of exposure to oleic acid, and exposure to oleic acid for as little as 15 minutes caused a subsequent decrease in LPL activity. LPL activity recovered 48 hours after removal of oleic acid from culture medium. Decreased LPL activity after oleic acid exposure was also noted in epididymal cells and in differentiated adipocyte precursors. The extent of decrease of LPL activity upon fatty acid exposure was dependent on the presence of the carboxyl group and was affected by acyl chain length. Although oleic acid did not affect protein synthesis estimated by [ 3H]-leucine incorporation, LPL mRNA levels were decreased following exposure of cells to oleic acid. Glycerol-3-phosphate dehydrogenase (G3PD) activity and mRNA levels were not affected by oleic acid exposure. Hence, fatty acids cause a dose-, acyl chain-, and carboxyl group-dependent specific decrease of heparin-releasable and intracellular LPL activities in cultured rat adipocyte precursors; this effect is associated with and is likely caused at least in part by a decrease in LPL mRNA levels.

Distribution and source of lipoprotein lipase in mouse mammary gland

During lactation lipoprotein lipase (LPL) is elevated in mammary tissue and depressed in adipose tissue to redirect lipids for incorporation into milk fat. The cellular origin of lipoprotein lipase in mammary tissue is thought to be the mammary epithelial cell which is the predominant cell type noticeable in the lactating gland; however, mammary adipocytes are also present. If lipoprotein lipase is produced by adipocytes in other sites of the body, then the question remains as to why mammary adipocytes have not been shown to produce lipoprotein lipase. In this study we present several lines of evidence that indicate that the mammary adipocyte is a source of LPL in the lactating mammary gland of mice. This evidence includes the absence of extracellular and intracellular lipoprotein lipase activity in two types of primary mammary epithelial cell cultures and a similarity in the changes of lipoprotein lipase activity in genital adipose tissue from nonpregnant mice and lactating mammary tissue to the nutritional state of the animal. Other evidence presented here includes strong localization of lipoprotein lipase protein and messenger RNA by fluorescence immunohistochemistry and in situ hybridization, respectively, to interstitial cells located between epithelial structures. We postulate that these interstitial cells are regressed, lipid-deleted mammary adipocytes.

Combined lipase deficiency in the mouse. Evidence of impaired lipase processing and secretion.

Newborn combined lipase-deficient (cld) mice have severe hypertriglyceridemia associated with a marked decrease of lipoprotein lipase (LPL) and hepatic lipase (HL) activities. Since the cld mutation and lipase genes reside on separate chromosomes, combined lipase deficiency cannot result from defects occurring within the LPL or HL structural genes. To elucidate the biochemical basis of this trans-acting defect, cld mice were compared to unaffected littermates for changes in lipase mRNA levels, rates of synthesis, and posttranslational processing and secretion. LPL and HL mRNA levels in cld liver and LPL in cld heart were comparable to controls; corresponding lipase synthetic rates were modestly decreased by about 30%. However, these reduced synthetic rates were not lipase-specific, since the rates of apolipoprotein (apo) A-I and apoA-II synthesis in cld liver were similarly decreased. Despite LPL synthetic rates that were 70% of controls, LPL mass in cld postheparin plasma was markedly reduced to only 7% of control values, suggesting that the majority of LPL is not secreted but remains intracellular. Consistent with a lipase secretory defect, neither the LPL nor HL oligomannosyl forms were converted to their respective complex forms in cld tissues, indicating that the lipases had failed to move from the endoplasmic reticulum/cis-Golgi to the medial/trans-Golgi network. In addition, the majority of intracellular LPL was catalytically inactive, since LPL specific activity (units/mg LPL protein) in cld heart, kidney, and brain was reduced 80-97%. In contrast to the severe impairment of lipase posttranslational processing and secretion, cld mouse plasma contained normal levels of another secretory N-linked glycoprotein, adipsin, with its oligosaccharide chains fully processed to the complex form. Thus, the cld mutation appears not to globally disrupt the secretion of all N-linked glycoproteins, but rather selectively impairs LPL and HL at points essential to their normal intracellular transport and secretion.

Biosynthesis of Lipoprotein Lipase in Cultured Mouse Adipocytes: II. Processing, subunit assembly, and intracellular transport

Abstract The biosynthesis and turnover of lipoprotein lipase (LPL) have been investigated in adipose 3T3-F442A cells labeled with [35S]methionine. Pulse-chase experiments, endo-beta-N-acetylglucosaminidase H treatment, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis have indicated that LPL is synthesized in the endoplasmic reticulum as a glycoprotein of Mr = 55,500 bearing two N-oligosaccharide side chains of the high mannose-type. This precursor form of LPL is transported within 10 min to the Golgi apparatus, and this event is accompanied by the formation of a mature species of Mr = 58,000. Treatment of the Mr = 58,000 species with glycopeptidase F yielded a Mr = 51,000 protein similar to that observed after treatment of the Mr = 55,500 precursor form or after inhibition of N-glycosylation in tunicamycin-treated cells. The precursor form of LPL of Mr = 55,500 does not accumulate in the cells since, after a labeling period of 2 h, only the Mr = 58,000 species is detected. It is shown that only 20% of the newly synthesized molecules of Mr = 58,000 are constitutively secreted, whereas 80% are degraded, most likely in lysosomes, as indicated by the inhibitory effect of leupeptin upon the degradation process. Under heparin stimulation, quantitative secretion of the mature form of LPL takes place whereas the intracellular degradation is arrested. Heparin is able to mobilize intracellular LPL without changing the rate of LPL export from the endoplasmic reticulum to the cell surface. Sucrose gradient centrifugation of the material from intracellular cisternae shows that the Mr = 55,500 precursor form is present as a monomer (s = 4.1 S), whereas the Mr = 58,000 mature form is present as a homodimer (s = 6.8 S) to which LPL activity is associated. The results are interpreted as LPL being transiently stored under a dimeric form before its degradation. A sorting process of LPL in the Golgi apparatus, followed by its entry either mainly in a regulated pathway or in a constitutive pathway, is proposed.

Comparison of lipoprotein lipase activity in heart myocytes and perfused hearts

This study was initiated to compare lipoprotein lipase activity in isolated heart myocytes and the heparin residual compartment of perfused hearts from adult rats. Heart lipoprotein lipase activity was divided into two fractions by 1 min of heparin perfusion. Heparin-residual and myocyte lipoprotein lipase activity were lower in hearts obtained from fasted compared to fed rats. In each case, the myocyte enzyme activity was 55 to 60% of heparin-residual levels. The difference between myocyte and heparin-residual activity may be a consequence of the time and treatment required to isolate cells in that long-term in vitro exposure of heart tissue to heparin also reduces residual activity. In vivo treatment with endotoxin decreased both heparin-residual and myocyte lipoprotein lipase activities; whereas, colchicine administration increased both activities compared to saline injected rats. In this latter experiment heart heparin-residual and myocyte lipoprotein lipase activities were positively correlated ( r = 0.90). The results indicate that in the mature heart intracellular lipoprotein lipase activity is primarily associated with myocytes.

Role of the alkaline TG lipase in regulating intramuscular TG content

Three TG lipases have been identified in muscle (i.e., acid, neutral, and alkaline), but as yet we do not know which enzyme is responsible for tissue TG hydrolysis. Over the past 8 yr, work in our laboratory has focused on intracellular lipoprotein lipase (LPL). The results show that this lipase is regulated by the classical cAMP cascade and that the activity of this enzyme is inversely related to endogenous TG concentration. Using these results as a foundation we plan to examine molecular mechanisms involved in the synthesis, compartmentalization, and transport of the alkaline TG lipase. Further, the evidence suggests that this enzyme may be regulated by protein phosphorylation mediated by cyclic AMP-dependent protein kinase. We plan to test this possibility.

Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes.

Lipoprotein lipase (LPL) is the enzyme responsible for hydrolysis of circulating triglyceride-rich lipoproteins and is important for storage of adipocyte lipid. To study the regulation of LPL synthetic rate in adipose tissue, primary cultures of isolated rat adipocytes were pulse-labeled with [35S]methionine, and LPL was immunoprecipitated with an LPL-specific antibody. A pulse-chase experiment identified the cellular and secreted forms of LPL as a 55-57-kDa protein. In the presence of heparin, there was a large increase in secretion of newly synthesized LPL from the cells, although heparin did not stimulate cellular LPL synthetic rate. When cells were exposed to insulin for 2 h, pulse-labeling revealed that insulin stimulated a maximal dose-related increase in LPL synthetic rate of 300% of control. This increase in LPL synthetic rate was observed after an exposure to insulin for as little as 60 min and was accompanied by only a 10-25% increase in total protein synthesis. In addition, insulin had no effect on the turnover of intracellular LPL. Using a cDNA probe for LPL, insulin induced a 2-fold increase in the LPL mRNA. Thus, insulin stimulated an increase in specific LPL mRNA in isolated rat adipocytes. This increase in LPL mRNA then leads to an increase in the synthetic rate of the LPL protein.

Importance of the different steps of glycosylation for the activity and secretion of lipoprotein lipase in rat preadipocytes studied with monensin and tunicamycin

Lipoprotein lipase synthesized by cultured rat preadipocytes is present in three compartments: an intracellular, a surface-related 3-min heparin-releasable, and that secreted into the culture medium. 30 min after addition of 6 μ M monensin, the lipoprotein lipase activity in the heparin-releasable compartment starts to decrease; by 4 h of monensin treatment the lipoprotein lipase activity in the heparin-releasable pool and in the culture medium is about 10% of that found in control dishes. The intracellular activity, which had been identified as lipoprotein lipase by an antiserum to lipoprotein lipase, increases slowly and doubles by 24 h. However, since the cellular compartment accounts for 10–25% of total activity, this increase does not account for the missing enzyme activity. To determine whether this enzyme molecule is synthesized but is not active, incorporation of labeled leucine, mannose and galactose into immunoadsorbable lipoprotein lipase was studied in control, monensin- or tunicamycin-treated cells. Addition of tunicamycin (5 μg/ml) for 24 h caused a 30–50% reduction in immunoadsorbable lipoprotein lipase, but the enzyme activity was reduced by 90%. On the other hand, 4 h monensin treatment reduced both incorporation of [ 3H]leucine into immunoadsorbable lipoprotein lipase and heparin-releasable and medium lipoprotein lipase activity by 57 to 77%. The immunoadsorbable lipoprotein lipase in the intracellular compartment has a [ 14C]mannose to [ 3H]galactose ratio of 0.15 and this ratio increased 6-fold in monensin-treated cells. The intracellular lipoprotein lipase in monensin-treated cells had the same affinity for both the native and synthetic substrate as the lipoprotein lipase in control cells, yet its spontaneous secretion into the culture medium and its release by 3 min heparin treatment was markedly decreased. The present results indicate that: (a) the presence of asparagine-linked oligosaccharide (formation of which is inhibited by tunicamycin) is mandatory for the expression of lipoprotein lipase activity; (b) lipoprotein lipase is active also in a high mannose form; and (c) terminal glycosylation and oligosaccharide processing, which is inhibited by monensin, may be important for the appearance of heparin-releasable lipoprotein lipase and secretion of lipoprotein lipase into the medium.

Relationship between type L hormone-sensitive lipase activity and endogenous triacylglycerol in the hearts of colchicine-treated rats.

Colchicine injection was used as a tool to potentiate the increase in intracellular lipoprotein lipase (type L hormone-sensitive lipase) activity normally seen with fasting to determine if elevation of enzyme activity by this method produced a reduction in endogenous triacylglycerol (TG) in rat heart. Both fasting and fasting+colchicine treatment increased total lipoprotein lipase (LPL) activity from a control value of 80 units/g to approx. 144 units/g. The initial control value was obtained at 08:00 h after overnight feeding and the final values were obtained at 17:00 h, after 9 h of fasting. Fasting alone increased activity in both the capillary-bound LPL and type L hormone-sensitive lipase (HSL) fractions of cardiac muscle. In contrast, colchicine treatment, by blocking the export of enzyme from the cell as a result of microtubular disruption, restricted the increase in enzyme activity to the intracellular fraction of the heart. There was a highly significant (P less than 0.001) negative relationship (r = -0.73) between type L HSL activity and TG content in hearts of fasting and fasting+colchicine-treated rats. At a time when type L HSL activity was increased and TG content decreased, the cyclic AMP concentration of heart remained unchanged, ruling out the possibility that cyclic AMP might be activating any one of the identified cardiac TG lipases. These data provided indirect evidence that type L HSL is 'seeing the intracellular TG droplet' and that this enzyme may play a role in the regulation of myocardial lipolysis.