The hormonal responses of lipoprotein lipase activity and lipolysis in adipose tissue differ depending on the stage of the estrous cycle in female rats.

OBJECTIVE: This study was designed to elucidate whether there were differences in the hormonal responses of the parameters involving triacylglycerol (TG) deposition and mobilization in adipose tissue among the stages of the estrous cycle in female rats. MEASUREMENTS: Adipose tissue was obtained from the parametrial region in female rats at each stage of the estrous cycle. Lipoprotein lipase (LPL) activity in the extracts of acetone/ether powders of the tissues was measured as a parameter for TG deposition. Norepinephrine-stimulated lipolysis in isolated fat cells was measured as a parameter for TG mobilization. RESULTS: LPL activity changed periodically during the estrous cycle; the activity level was highest at diestrus, began to decrease at proestrus, reached a minimum at estrus, began to increase again at metestrus-1, and increased further at metestrus-2. At diestrus and proestrus, LPL activity was increased with an increase in plasma insulin levels, suggesting that plasma insulin was the predominant up-regulator of LPL. But at estrus, metestrus-1 and metestrus-2, LPL activity remained low even when plasma insulin levels were high, indicating that it was not up-regulated by plasma insulin. Norepinephrine-stimulated lipolysis in fat cells was high at estrus and metestrus-1 and low at diestrus. CONCLUSION: The hormonal responses of LPL activity and lipolysis in adipose tissue differed depending on the stage of the estrous cycle.

Compositional analysis of root cementum and dentin after Er:YAG laser irradiation compared with CO2 lased and intact roots using Fourier transformed infrared spectroscopy.

The present study examines the dental root after Er:YAG laser irradiation, compared with CO2 lased and non-treated surfaces, using Fourier Transformed Infrared (FTIR) spectroscopy. Freshly extracted human teeth were irradiated by Er:YAG laser at an energy output of 40 mJ/pulse, 10 Hz (0.4 watts), with or without water coolant, and by CO2 laser at an energy output of 0.5 watts in continuous wave mode without coolant. The surfaces were chalky and smooth after irradiation by Er:YAG laser with water coolant, were charred and irregular after irradiation by Er:YAG laser without water coolant, and were completely carbonized after CO2 laser irradiation. The FTIR profiles from samples of the surfaces that were irradiated by Er:YAG laser with water coolant were similar to those from non-treated samples, except for a slight decrease on the OH and amide bands, which are mainly related to organic components. This decrease was observed to be extreme after CO2 laser irradiation and moderate after Er:YAG laser irradiation without coolant. The formation of new bands showing toxic substances was observed to a large extent after CO2 laser irradiation and to a smaller extent after Er:YAG laser irradiation without water coolant. In contrast, no such bands were detected after Er:YAG laser irradiation with water coolant. The present results show that these laser treatments selectively ablated more organic components than inorganic components and that Er:YAG laser irradiation with water coolant did not cause major compositional changes or chemically deleterious changes in either root cementum or dentin.

Ligand recognition by the vitamin D receptor.

Three-dimensional structure of the ligand binding domain (LBD) of the vitamin D receptor (VDR) docked with the natural ligand 1 alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] has been mostly solved by the X-ray crystallographic analysis of the deletion mutant (VDR-LBD Delta 165-215). The important focus, from now on, is how the VDR recognizes and interacts with potent synthetic ligands. We now report the docking models of the VDR with three functionally and structurally interesting ligands, 22-oxa-1,25-(OH)(2)D(3) (OCT), 20-epi-1,25-(OH)(2)D(3) and 20-epi-22-oxa-24,26,27-trihomo-1,25-(OH)(2)D(3). In parallel with the computational docking studies, we prepared twelve one-point mutants of amino acid residues lining the ligand binding pocket of the VDR and examined their transactivation potency induced by 1,25-(OH)(2)D(3) and these synthetic ligands. The results indicate that L233, R274, W286, H397 and Y401 are essential for holding the all ligands tested, S278 and Q400 are not important at all, and the importance of S237, V234, S275, C288 and H305 is variable depending on the side-chain structure of the ligands. Based on these studies, we suggested key structural factors to bestow the selective action on OCT and the augmented activities on 20-epi-ligands. Furthermore, the docking models coincided well with our proposed active space-region theory of vitamin D based on the conformational analyses of ligands.

Three-dimensional structure-function relationship of vitamin D and vitamin D receptor model.

On the basis of conformational analysis of the vitamin D side chain and studies using conformationally restricted synthetic vitamin D analogs, we have suggested the active space region concept of vitamin D: The vitamin D side-chain region was grouped into four regions (A, G, EA and EG) and the A and EA regions were suggested to be important for vitamin D actions. We extended our theory to known highly potent vitamin D analogs and found a new region F. The analogs which occupy the F region have such modifications as 22-oxa, 22-ene, 16-ene and 18-nor. Altogether, the following relationship between the space region and activity was found: Affinity for vitamin D receptor (VDR), EA > A> F > G > EG; Affinity for vitamin D binding protein (DBP), A >> G,EA,EG; Target gene transactivation, EA > F > A > EG > or = G; Cell differentiation, EA > F > A > EG > or = G; Bone calcium mobilization, EA > GA > F > or = EG; Intestinal calcium absorption, EA = A > or = G >> EG. We modeled the 3D structure of VDR-LBD (ligand binding domain) using hRARgamma as a template, to develop our structure-function theory into a theory involving VDR. 1alpha,25(OH)(2)D(3) was docked into the ligand binding pocket of the VDR with the side chain heading the wide cavity at the H-11 site, the A-ring toward the narrow beta-turn site, and the beta-face of the CD ring facing H3. Amino acid residues forming hydrogen bonds with the 1alpha- and 25-OH groups were specified: S237 and R274 forming a pincer type hydrogen-bond for the 1alpha-OH and H397 for the 25-OH. Mutants of several amino acid residues that are hydrogen-bond candidates were prepared and their biologic properties were evaluated. All of our mutation results together with known mutation data support our VDR model docked with the natural ligand.

Synthesis of active high mannose-type lipoprotein lipase in human adipose tissues.

Lipoprotein lipase (LPL) activity in the retroperitoneal adipose tissue of a patient with Cushing's syndrome and in the subcutaneous adipose tissue of a patient with aseptic necrosis of the femoral head was higher than that in the corresponding tissues of the control subjects. The amount of [35S]methionine incorporated into LPL was also higher in these patients than in control subjects. However, the ratio of activity and amount of radioactivity in the LPL of patients was identical to that of control subjects, indicating that LPL synthesized in the adipose tissues of patients had a normal specific activity. LPL with Mr = 57000 was composed of two types of subunits: one type was partially endo H-sensitive, yielding a product with Mr = 55000, and the other was totally endo H-sensitive, yielding a product with Mr = 52000. Both retroperitoneal and subcutaneous adipose tissues of control subjects contained nearly equal amounts of partially and totally endo H-sensitive subunits. In the retroperitoneal adipose tissue of a patient with Cushing's syndrome, 8% of subunits were partially endo H-sensitive and 92% were totally endo H-sensitive. In the subcutaneous adipose tissue of a patient with aseptic necrosis of the femoral head, 21% of subunits were partially endo H-sensitive and 79% were totally endo H-sensitive. The 24-h treatment of subcutaneous adipose tissue of a control subject with 1 mM 1-deoxymannojirimycin (dMM) caused the synthesis of active, but totally endo H-sensitive, LPL. Thus, in human adipose tissue, the processing of one oligosaccharide chain of an LPL subunit to a complex type chain in the trans Golgi was not necessary for the expression of activity.

Structure-function analysis of vitamin D and VDR model.

In the first section, the general three-dimensional structure of the ligand-binding domain (LBD) of nuclear receptors (NR) was briefly described on the basis of their x-ray crystal structures. Emphasis was placed on the three major conformations of NR-LBD and their role in the transactivation function. In the second part, the structure-function relationship of vitamin D was analyzed based on the ligand structure, in particular by using systematic conformational analysis as a tool. On the basis of the conformational analysis of the vitamin D side chain and studies using conformationally restricted synthetic vitamin D analogs, we suggested the active space region concept of vitamin D: The vitamin D side-chain region was grouped into five regions (A, G, EA, EG and F). Activity orders, in terms of the spatial region, found by these studies are as follows: Affinity for vitamin D receptor (VDR), EA>A>F>G>EG; Affinity for vitamin D binding protein (DBP), A>>G,EA, EG; Target gene transactivation, EA>F>A>EG G; Cell differentiation, EA>F>A>EG G; Bone calcium mobilization, EA>G A>F EG; Intestinal calcium absorption, EA=A G>>EG. In the third section, homology modeling of VDR-LBD and docking of the natural ligand, 1,25-(OH)2D3, into the ligand binding cavity of the model are described. Amino acid residues forming hydrogen bonds with the biologically important 1alpha- and 25-OH groups were identified: 1alpha-OH forms a pincer-type hydrogen bond with R274 and S237 and 25-OH with H397. This VDR-LBD/1,25-(OH)2D3 docking model was firmly substantiated by mutation analysis. Using this VDR model, the structure-function relationship of highly potent vitamin D analogs was discussed.

Three-dimensional modeling of and ligand docking to vitamin D receptor ligand binding domain.

The ligand binding domain of the human vitamin D receptor (VDR) was modeled based on the crystal structure of the retinoic acid receptor. The ligand binding pocket of our VDR model is spacious at the helix 11 site and confined at the beta-turn site. The ligand 1alpha, 25-dihydroxyvitamin D(3) was assumed to be anchored in the ligand binding pocket with its side chain heading to helix 11 (site 2) and the A-ring toward the beta-turn (site 1). Three residues forming hydrogen bonds with the functionally important 1alpha- and 25-hydroxyl groups of 1alpha,25-dihydroxyvitamin D(3) were identified and confirmed by mutational analysis: the 1alpha-hydroxyl group is forming pincer-type hydrogen bonds with S237 and R274 and the 25-hydroxyl group is interacting with H397. Docking potential for various ligands to the VDR model was examined, and the results are in good agreement with our previous three-dimensional structure-function theory.

Butyltin residues in livers of humans and wild terrestrial mammals and in plastic products.

Butyltin compounds (BTs) including mono-(MBT), di-(DBT) and tributyltin (TBT) were determined in livers of humans and wild terrestrial mammals, such as raccoon dogs (Nyctereutes procyonoids) and monkeys (Macaca fuscata) from Japan. In addition, 22 samples of plastic products were analyzed. BT residues were detected in all the liver samples of humans and raccoon dogs, with concentrations of <360 ng/g wet wt, whereas concentrations in the liver of monkeys were either less than the detection limit or were only in trace levels. Elevated concentrations of BTs, particularly DBT (<140,000 ng/g) and MBT (<130,000 ng/g), were found in some plastic products, such as baking parchments made from siliconized paper and gloves made up from polyurethane. The results of a cooking test using the above baking parchment indicated the transfer of BTs to foodstuffs. These observations suggest expansion of BT contamination among terrestrial mammals. BT pollution from industrial appliances, such as plastic stabilizers and catalysts other than those of marine origin as antifouling agents, are suggested as alternative sources of exposure.

Conformation-function relationship of vitamin D: conformational analysis predicts potential side-chain structure.

In previous studies, we have grouped regions in space occupied by the vitamin D side chain into four: A, G, EA, and EG. We showed that the receptor (VDR) affinity of 1alpha,25-dihydroxyvitamin D3 derivatives increases, in terms of side-chain region, in the order EG, G, A, and EA. We called this the active space group concept. In the present study, we used this active space group concept to analyze the conformation-activity relationship of about 40 representative potent 1alpha,25-dihydroxyvitamin D3 analogues. We initially listed structural modifications in the side chain of potent vitamin D analogues and estimated their potency factor. Possible side-chain conformations of representative analogues were calculated by the molecular mechanics method and plotted on a dot map compared with the regions A, G, EA, and EG. The cell-differentiating potency of the analogues was correlated with our active space group concept with few exceptions. Among potent analogues with a natural configuration at C(20), the side chains of those with a 22-oxa, 22-ene, 16-ene, or a 18-nor modification were located in front of region EA (termed F). The side chains of the most potent 20-epi-22-oxa-24-homovitamin D analogues were concentrated at the left side of the EA region (L-EA). Thus, the side chains of almost all potent analogues were distributed around the EA region, and potency increased in the order A, F, EA, and L-EA.

Effect of long-term treatment of 3T3-L1 adipocytes with chlorate on the synthesis, glycosylation, intracellular transport and secretion of lipoprotein lipase.

Lipoprotein lipase (LPL) is synthesized and glycosylated in the endoplasmic reticulum (ER), transported through the Golgi to the cell surface, and finally secreted. To examine the role of heparan sulphate proteoglycans (HSPG) in the synthesis, activity, intracellular transport and secretion of LPL, 3T3-L1 adipocytes were cultured for 7 days in the presence of 20 mM chlorate, an inhibitor of sulphation of HSPG. Treatment of cells with 20 mM chlorate for 7 days caused a 55% decrease in LPL activity in the intracellular compartment and a 79% decrease in the cell-surface compartment. The synthetic rate of LPL in chlorate-treated cells was identical with that in control cells as determined by biosynthetic labelling. The study with endoglycosidase H (endo H) showed that the treatment with chlorate increased the proportion of LPL subunits which were totally endo H-sensitive. The study with a heparin-Sepharose column showed that 3T3-L1 adipocytes contained three forms of LPL. The first form, accounting for 35% of the LPL, did not bind to the heparin-Sepharose column and had little or no activity; the second form, accounting for 32%, bound to the column and was eluted with 0.4-0.75 M NaCl but had no activity; the third form, accounting for 33%, bound to the column and was eluted with 0.8-1.2 M NaCl and had activity. In chlorate-treated cells, the first form accounted for 66% of the LPL, the second form 15% and the third form 19%. When cells were incubated for 1 h with brefeldin A, which translocates Golgi proteins to the ER [J. Lippincott-Schwartz, L.C. Yuan, J.S. Banifacino and R.D. Klausner (1989) Cell 56, 801-813; J. Lippincott-Schwartz, J. Glickman, J.E. Donaldson, J. Robbins, T.E. Kreis, K.B. Seamon, M.P. Sheetz and R.D. Klausner (1991) J. Cell Biol. 112, 567-577], the chlorate-induced decrease in cellular LPL activity was restored. These findings indicate that LPL synthesized in chlorate-treated cells can be processed to be fully active, but chlorate-treated cells are unable to transport LPL to the Golgi and accumulate inactive LPL with a lower affinity for heparin in the ER. The treatment with chlorate decreased the proportion of LPL subunits that were endo H-resistant, indicating that the processing of oligosaccharide chains of LPL in the trans-Golgi was impaired in chlorate-treated cells. The amount of 35S-labelled LPL secreted by chlorate-treated cells was identical with that secreted by control cells, whereas the level of LPL activity in the medium of chlorate-treated cells was 25% of that in the medium of control cells, indicating that most of the LPL secreted by chlorate-treated cells was inactive.

Ginsenosides increase secretion of lipoprotein lipase by 3T3-L1 adipocytes.

Treatment of 3T3-L1 adipocytes with either an oleanolic acid glycoside or a 20(S)-protopanaxatriol glycoside increased the secretion of lipoprotein lipase activity into the medium dose-dependently. At a concentration of 100 micrograms/ml, ginsenosides Ro, Re, Rg1, and Rh1 increased the secretion of lipase activity into the medium by 119, 107, 56, and 32%, respectively. The ratio of lipase activity in the medium to cellular lipase activity was 4.7% in control cells and 8.6% in ginsenoside Ro-treated cells, 8.3% in ginsenoside Re-treated cells, 7.0% in ginsenoside Rg1-treated cells, and 6.3% in ginsenoside Rh1-treated cells. Ginsenoside Rb2, which is a 20(S)-protopanaxadiol glycoside, increased the secretion of lipase activity by 16% at 25 micrograms/ml, and the ratio of lipase activity in the medium to cellular lipase activity was higher in ginsenoside Rb2-treated cells than in control cells. However, at 100 and 200 micrograms/ml, ginsenoside Rb2 decreased the secretion of lipase activity in parallel with cellular lipase activity. Ginsenoside Rd also decreased the secretion of lipase activity in the same dose-dependent manner. Thus, the effective dose for the secretion of lipoprotein lipase activity with ginsenosides varies with their aglycone structure.

Recombinant human tumour necrosis factor-alpha suppresses synthesis, activity and secretion of lipoprotein lipase in cultures of a human osteosarcoma cell line.

The effect of recombinant human tumour necrosis factor-alpha (TNF-alpha) on synthesis, activity and secretion of lipoprotein lipase (LPL) was examined using a human osteosarcoma cell line, osteosarcoma Takase (OST). Treatment of OST cells with TNF-alpha decreased LPL synthesis, resulting in a decrease in expression of activity and secretion of LPL. When OST cells were incubated with glycerol tri[1-14C]palmitate, TNF-alpha decreased dose- and time-dependently the production of 14CO2 and the amounts of radioactivity incorporated into cellular triacylglycerol and phospholipid. The similar reduction of synthesis and activity of LPL as suppression of CO2 production and cellular lipid synthesis indicated that the suppression of 14CO2 production and 14C-labelled lipid synthesis was secondary. TNF-alpha also suppressed expression of proliferating cell nuclear antigen, indicating that it had an anti-proliferative activity on OST cells. The findings suggest that one cause of the anti-proliferative activity of TNF-alpha is the suppression of the LPL-mediated supply of non-esterified fatty acids as an energy source for growth.

Reduced dimerization of lipoprotein lipase in post-heparin plasma of a patient with hyperchylomicronemia.

As in post-heparin plasma of control subjects, post-heparin plasma of a patient with hyperchylomicronemia contained lipoprotein lipase (LPL) subunits with M(r) = 57,000. But although the amount of LPL was the same as in post-heparin plasma of controls, no LPL activity was detectable. Nearly all the LPL in post-heparin plasma of controls bound to heparin-Sepharose and this LPL bound was mainly eluted with 1.5 M NaCl in parallel with the activity. In post-heparin plasma of the patient, 58% of the LPL subunits did not bind to heparin-Sepharose and 23% was eluted with 0.6 M NaCl. Studies by sucrose density gradient centrifugation showed that almost all the LPL in post-heparin plasma of controls was recovered in the peak with a sedimentation coefficient of 6.8 S, corresponding to the position of a dimeric form of LPL, in parallel with the activity; little LPL was recovered in the peak with a sedimentation coefficient of 4.0 S, corresponding to the position of a monomeric form of LPL. In post-heparin plasma of the patient, 35% of the LPL subunits was recovered in fractions with larger sedimentation coefficients at the bottom of the centrifuge tube, indicating the presence of an aggregated form(s) of LPL; the amount of the monomeric form of LPL was increased, while that of the dimeric form was decreased. Thus, defect of LPL activity in post-heparin plasma of the patient with hyperchylomicronemia could result from reduced dimerization of LPL subunits.

Glycosylation and secretion of lipoprotein lipase by 3T3-L1 adipocytes: effects of brefeldin A.

Time courses of synthesis and secretion of lipoprotein lipase (LPL) were examined in 3T3-L1 adipocytes. LPL was glycosylated in the endoplasmic reticulum (ER) within 10 min after synthesis, and was transported after 20-30 min to the trans Golgi where it was converted to the mature form with M(r) = 55,000-58,000, which was resistant to endoglycosidase H (endo H). LPL subunits with M(r) = 55,000-58,000 appeared in the medium within 30 min after synthesis. The effects of brefeldin A (BFA), which inhibits transport of glycoproteins in various types of cells, on secretion and glycosylation of LPL were also examined. BFA completely blocked release of LPL activity into the medium, causing accumulation of the activity in cells. The suppressive effect of BFA on release of LPL activity was reversible. BFA-treated cells synthesized LPL with M(r) = 53,000-55,000 consisting of 2 types of subunits, the main type being totally endo H-sensitive and the other partially endo H-sensitive. No LPL were secreted into the medium by BFA-treated cells.

Dexamethasone induces the GLUT4 glucose transporter, and responses of glucose transport to norepinephrine and insulin in primary cultures of brown adipocytes.

Glucose uptake into brown adipose tissue is enhanced directly by norepinephrine released from the sympathetic nerves. In the present study, we tried to establish culture conditions for brown adipocytes which are favorable for investigation of this unique glucose transport. Stromal-vascular cells isolated from the interscapular brown adipose tissue of newborn rats differentiated into brown adipocytes expressing the uncoupling protein, when the cells were maintained on collagen-coated dishes. These cells, however, did not show an increase in 2-[3H]deoxyglucose transport in response to insulin or norepinephrine, nor did they exhibit expression of the GLUT4 glucose transporter, whereas GLUT1 was present, as judged on Western blotting. Pre-treatment of confluent cells with dexamethasone induced a response of glucose transport to either insulin or norepinephrine, and the expression of GLUT4, together with notable accumulation of lipid droplets. The induction of GLUT4 expression by dexamethasone was dose-dependent and potentiated by insulin. These results indicate that treatment of cultured brown adipocytes with dexamethasone makes it feasible to analyze the mechanism underlying the enhancement of glucose transport induced by norepinephrine.

Existence of lipoprotein lipase in human sarcomas and carcinomas.

Aqueous extracts of acetone/ether powders of surgically obtained specimens of human tumors hydrolyzed 3H-labeled triolein in a dose-dependent manner. The lipolytic activity in these extracts was inhibited by anti-lipoprotein lipase (LPL) IgG dose-dependently, 25 micrograms of anti-LPL IgG causing 95% inhibition of the activity. Thus, LPL accounts for most of the lipolytic activity in extracts of acetone/ether powders of the tumors. All sarcomas and carcinomas examined contained LPL activity. Western blotting showed that they gave a band corresponding to that of human adipose tissue LPL (M(r) = 57,000). Immunocytochemical studies showed that LPL was present in cultured human osteosarcoma cells and distributed throughout the cells. We determined the proliferating cell nuclear antigen (PCNA)-labeling index as an indicator of the proliferative activity of tumor cells and measured LPL activity in extracts of tumors in areas corresponding to those used for determining the PCNA-labeling index. In malignant fibrous histiocytomas, the PCNA-labeling index in area a, which corresponds to the subcapsular region, was higher than that in area b, which corresponds to the central region. The LPL activity in area a was 10 times that in area b. In rectal cancer, the index in area c, which corresponds to the subserosal region, was higher than that in area d, which corresponds to the submucosal region. The LPL activity in area c was 1.9 times that in area d. These findings indicate heterogeneity in the distributions of LPL activity within tumors and higher levels of LPL activity in tumors that are proliferating actively.

Role of processing of the oligosaccharide chains in the affinity of lipoprotein lipase for heparin.

The role of processing of the oligosaccharide chains in the affinity of lipoprotein lipase (LPL) for heparin was examined in 3T3-L1 adipocytes. 43% of 35S-labeled LPL subunits in tunicamycin (TUN)-treated cells did not bind to a heparin-Sepharose column and 46% was eluted with 0.6 M NaCl. 11% of LPL subunits in castanospermine (CSTP)-treated cells did not bind to the column and 38% was eluted with 0.6 M NaCl. In contrast, as in untreated cells, LPL subunits in 1-deoxymannojirimycin (dMM)-treated and swainsonine (SW)-treated cells almost all bound to the column and over 93% of the subunits bound were eluted with 1.5 M NaCl. Thus, core glycosylation and subsequent removal of the distal glucose residue from oligosaccharide chains of LPL in the endoplasmic reticulum (ER) is required for acquisition of a higher affinity for heparin.

Lipid droplet accumulation and lipoprotein lipase activity in the rat salivary gland during the perinatal period.

The submandibular and sublingual glands of foetal and newborn rats aged 21 days in utero to 7 days after birth were examined morphologically and biochemically. Lipid droplets tended to be localized in secretory cells, especially in their basal cytoplasm. The degree of droplet accumulation varied with the age of the rat. No droplets were observed before and immediately after birth. The number of accumulated droplets peaked 24-48 h after birth, then gradually decreased and reached normal levels by 5 days. In the salivary glands of fasted newborn rats, no lipid droplets were observed throughout the experiment. The amount of triacylglycerol reached its maximum level 1 day after birth; it then decreased gradually until 5 days and after that did not change. The amount of cholesterol did not change during postnatal development. Lipase activity attained its maximum level in the salivary glands immediately after birth and then decreased rapidly. It was higher in the glands of fasted than fed 1-day-old rats. Antiserum against lipoprotein lipase inhibited the salivary gland lipase activity in a dose-dependent manner, with 5 microliters of antiserum producing 60-70% inhibition. Non-immune serum had little effect. It was concluded that (1) accumulated lipid in the secretory cell cytoplasm of the salivary glands originates from ingested milk; (2) the principal component of accumulated lipid droplets is triacylglycerol; (3) 60-70% of the total lipase activity represents lipoprotein lipase; (4) an increase of lipoprotein lipase activity is recognizable before the accumulation of triacylglycerol.

Retention of glucose by N-linked oligosaccharide chains impedes expression of lipoprotein lipase activity: effect of castanospermine.

The effect of castanospermine (CSTP), an inhibitor of glucosidase I, on processing, activity, and secretion of lipoprotein lipase was studied in 3T3-L1 adipocytes. Processing was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of endoglycosidase H (endo H)-digested subunits of lipoprotein lipase from cells incubated 1-2 h with [35S]methionine. Lipoprotein lipase in untreated cells consisted of two groups of subunits, M(r) = 55,000-58,000 and M(r) = 53,000-55,000. The heavier subunits were endo H-resistant, whereas the others were either totally or partially endo H-sensitive. The lipase secreted by untreated cells contained primarily endo H-resistant subunits. Immunofluorescent studies showed that lipoprotein lipase accumulated in Golgi in untreated cells. CSTP, 100 micrograms/ml for 18 h, decreased intracellular lipase activity by 80% and decreased secretion of lipase activity by 91%. Most of the lipase subunits in CSTP-treated cells were totally endo H-sensitive with M(r) = 57,000, some were partially endo H-sensitive, and a trace was endo-H resistant. Totally endo H-sensitive subunits in CSTP-treated cells had a M(r) 2,000-4,000 larger than that in untreated cells, indicating impaired trimming of sugar residues from oligosaccharide chains of the lipase in CSTP-treated cells. The small amount of lipase secreted by CSTP-treated cells consisted primarily of partially endo H-sensitive subunits, with one sensitive and one resistant chain per subunit. Immunofluorescent studies showed that lipoprotein lipase was excluded from Golgi in CSTP-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)