From a total of 22 hypertriglyceridemic subjects tested, 14 subjects were selected on the basis of normal postheparin plasma lipoprotein lipase (LPL) levels and the presence of LPL inhibitory activity in their fasting plasma. The inhibitory activity was detected in both the lipoprotein fraction (d < 1.25 g/ml) and the lipo~protein-deficient fraction (d > 1.25 g/ml). Correlational analyses of LPL inhibitory activity and apolipoprotein levels present in the lipoprotein fraction (d < 1.25 g/ml)indicated that only apolipoprotein C-III (ApoC-III) was significantly correlated (r = 0.602, P < 0.05) with the inhibition activity of the lipoprotein fraction. Furthermore, it was found that LPL-inhibitory activities of the plasma lipoprotein fraction and lipoprotein-deficient fraction were also correlated (r = 0.745, P < 0.005), though the activity in the lipoprotein-deficient plasma was not related to the ApoC-III or apolipoprotein E levels. Additional correlational analyses indicated that the LPL levels in the postheparin plasma of these subjects were inversely related to the levels of plasma apolipoproteins C-II, C-III, and E. To explain some of these observations, we directly examined the in vitro effect of ApoC-III on LPL activity. The addition of ApoC-III-2 resulted in a decreased rate of lipolysis of human' very low density lipoproteins by LPL. Kinetic analyses indicated that ApoC-III-2 was a noncompetitive inhibitor of LPL suggesting a direct interaction of the inhibitor with LPL. Results of these studies suggest that ApoCIII may represent a physiologic modulator of LPL activity levels and that the incidence of LPL inhibitory activity in the plasma of hypertriglyceridemic subjects is more common than previously recognized.

Bile‐salt activated lipase (BAL) is a páncreatic enzyme that digests a variety of lipids in the small intestine. A distinct property of BAL is its dependency on bile salts in hydrolyzing substrates of long acyl chains or bulky alcoholic motifs. A crystal structure of the catalytic domain of human BAL (residues 1‐538) with two surface mutations (N186D and A298D), which were introduced in attempting to facilitate crystallization, has been determined at 2.3 Å resolution. The crystal form belongs to space group P2 1 2 1 2 1 with one monomer per asymmetric unit, and the protein shows an α/β hydrolase fold. In the absence of bound bile salt molecules, the protein possesses a preformed catalytic triad and a functional oxyanion hole. Several surface loops around the active site are mobile, including two loops potentially involved in substrate binding (residues 115‐125 and 270‐285).

We have expressed and purified a truncated recombinant human milk bile salt-activated lipase (T-BAL) from the T7 expression system in Escherichia coli. This T-BAL contains the N-terminal 538 residues of the 722-residue native enzyme. The purified T-BAL, when assayed with PANA (p-nitrophenyl acetate), had a specific activity of 64 +/- 2 units/mg (n = 4), as compared to 52 units/mg for the native enzyme. Because the recombinant T-BAL expressed in E. coli is not glycosylated, these results indicated that the highly glycosylated C-terminal region of BAL is not essential for catalytic function. Heat inactivation patterns of native BAL and T-BAL were found to be similar, further suggesting that the folding of T-BAL is similar to that of the catalytic domain of the native enzyme. With the availability of a sufficient amount of recombinant T-BAL, the specificity and kinetics of T-BAL and native BAL were compared. Fluorescence studies of T-BAL indicated that it has a slightly higher affinity for the monomeric form of taurocholate with a dissociation constant (KA) of 0.32 mM, compared with the reported 0.37 mM for the native enzyme. Further kinetic analysis indicated that there are enzyme specificity changes revealed with the use of PANA and PANB (p-nitrophenyl butyrate) as substrates. When assayed in the presence of taurocholate, T-BAL has a higher turnover rate constant with p-nitrophenyl acetate than with p-nitrophenyl butyrate, which was found to be in contrast to native BAL.(ABSTRACT TRUNCATED AT 250 WORDS)

The bone marrow stroma consists of a heterogeneous population of cells which participate in osteogenic, adipogenic, and hematopoietic events. The murine stromal cell line, BMS2, exhibits the adipocytic and osteoblastic phenotypes in vitro. BMS2 differentiation was examined in response to cytokines which share the gp130 signal transducing protein within their receptor complex. Four of the cytokines (interleukin 6, interleukin 11, leukemia inhibitory factor, and oncostatin M) inhibited hydrocortisone‐induced adipocyte differentiation in a dose dependent manner based on lipid accumulation and lipoprotein lipase enzyme activity. Inhibition occurred only when the cytokines were present during the initial 24 h of the induction period; after 48 h, their effects were diminished. Likewise, these cytokines increased alkaline phosphatase enzyme activity twofold in preadipocyte BMS2 cells. Both leukemia inhibitory factor and oncostatin M induced early active gene expression in resting preadipocyte BMS2 cells and decreased the steady state mRNA level of a unique osteoblastic gene marker, osteocalcin. A fifth cytokine whose receptor complex shares the gp130 protein, ciliary neurotrophic factor, did not significantly regulate stromal cell differentiation when added by itself. However, with the addition of a missing component of its receptor complex, ciliary neurotrophic factor receptor α protein, this cytokine also inhibited BMS2 adipogenesis. Together, these data indicate that the cytokines whose receptors share the gp130 protein can modulate stromal cell commitment to the adipocyte and osteoblast differentiation pathways.

Preliminary work has shown that select triacylglycerols (TAGs) are upregulated in a preclinical model of MGD, suggesting that TAGs may be an important outcome variable in research involving human meibomian gland epithelial cells (HMGECs). The purpose of this study was to explore the HMGEC TAG lipidome in culture conditions known to influence differentiation. HMGECs were differentiated in DMEM/F12 with 10 ng/ml EGF, FBS (2% or 10%), and rosiglitazone (0, 20, or 50 μM) for two or five days. Following culture, lipids were extracted, processed, and directly infused into a Triple TOF 5600 mass spectrometer (SCIEX, Framingham, MA) with electrospray ionization. MS and MS/MSALL spectra were acquired in the positive ion mode and performed with the SWATH technology. Only the TAGs that were present in all 48 samples were included in the analysis. Multiple regression techniques were utilized to assess the effects of each factor (FBS, rosiglitazone, and culture duration) on each expressed TAG. The HMGEC TAG lipidome consisted of 115 TAGs with 42–62 carbons and zero to 10 double bonds. Fatty acyl chains had 14 to 26 carbons and zero to five double bonds. C18:1 (oleic acid, 25/115, 21.7%) and C16:0 (palmitic acid, 16/115, 13.9%) were the most common fatty acids. FBS, rosiglitazone, and culture duration were significant predictors for 93 TAGs (80.9%) with R2 values ranging from 0.20 to 0.77 (p < 0.05). FBS and rosiglitazone achieved significance (p < 0.05) for 80 (69.6%) and 67 TAGs (58.3%), respectively. Rosiglitazone demonstrated a selective upregulation of TAGs containing 16 or 18 carbons. Culture duration reached significance (p < 0.05) for only 36 TAGs (31.3%). When comparing the 10 most abundant C18:1-containing TAGs in meibum, FBS was a negative predictor for five TAGs (mean standardized coefficient [SC] = −0.58, p < 0.001), rosiglitazone was a positive predictor for six TAGs (mean SC = 0.41, p ≤ 0.03), and culture duration weakly influenced one TAG (SC = 0.27, p = 0.008). FBS and rosiglitazone, unlike culture duration, are powerful modulators of the TAG profile. Rosiglitazone induces changes that could be consistent with fatty acid synthesis, suggesting that quantifying the TAG lipidome could be an indirect measure of lipogenesis. Though both have been described as differentiating agents, FBS and rosiglitazone induce opposing effects on meibum-relevant TAGs. Culturing with rosiglitazone is associated with a TAG profile that is more consistent with the expected outcome of lipogenesis and with the profile observed in normal human meibum.

Journal Article Killing of Giardia lamblia Trophozoites by Human Intestinal Fluid in Vitro Get access Siddhartha Das, Siddhartha Das Departments of Pathology and of Medicine (Divisions of Infectious Diseases and of Gastroenterology), University of California, San Diego Medical Center, San Diego, California; and the Laboratory of Lipid and Lipoprotein Research, Oklahoma Medical Research, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma Search for other works by this author on: Oxford Academic PubMed Google Scholar David S. Reiner, David S. Reiner Departments of Pathology and of Medicine (Divisions of Infectious Diseases and of Gastroenterology), University of California, San Diego Medical Center, San Diego, California; and the Laboratory of Lipid and Lipoprotein Research, Oklahoma Medical Research, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma Search for other works by this author on: Oxford Academic PubMed Google Scholar John Zenian, John Zenian Departments of Pathology and of Medicine (Divisions of Infectious Diseases and of Gastroenterology), University of California, San Diego Medical Center, San Diego, California; and the Laboratory of Lipid and Lipoprotein Research, Oklahoma Medical Research, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma Search for other works by this author on: Oxford Academic PubMed Google Scholar Daniel L. Hogan, Daniel L. Hogan Departments of Pathology and of Medicine (Divisions of Infectious Diseases and of Gastroenterology), University of California, San Diego Medical Center, San Diego, California; and the Laboratory of Lipid and Lipoprotein Research, Oklahoma Medical Research, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma Search for other works by this author on: Oxford Academic PubMed Google Scholar Michael A. Koss, Michael A. Koss Departments of Pathology and of Medicine (Divisions of Infectious Diseases and of Gastroenterology), University of California, San Diego Medical Center, San Diego, California; and the Laboratory of Lipid and Lipoprotein Research, Oklahoma Medical Research, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma Search for other works by this author on: Oxford Academic PubMed Google Scholar Chi-Sun Wang, Chi-Sun Wang Departments of Pathology and of Medicine (Divisions of Infectious Diseases and of Gastroenterology), University of California, San Diego Medical Center, San Diego, California; and the Laboratory of Lipid and Lipoprotein Research, Oklahoma Medical Research, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma Search for other works by this author on: Oxford Academic PubMed Google Scholar Frances D. Gillin Frances D. Gillin Departments of Pathology and of Medicine (Divisions of Infectious Diseases and of Gastroenterology), University of California, San Diego Medical Center, San Diego, California; and the Laboratory of Lipid and Lipoprotein Research, Oklahoma Medical Research, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma Please address requests for reprints to Dr. F. D. Gillin, Department of Pathology H811F, University of California, San Diego Medical Center, 225 Dickson Street, San Diego, California 92103. Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 157, Issue 6, June 1988, Pages 1257–1260, https://doi.org/10.1093/infdis/157.6.1257 Published: 01 June 1988 Article history Received: 22 September 1987 Revision received: 28 December 1987 Published: 01 June 1988

A procedure for the purification of carboxyl ester lipase from human pancreas has been developed. The determined N-terminal 10 amino acid residues of the purified enzyme, NH2-Ala-Lys-Leu-Gly-Ala-Val-Tyr-Thr-Glu-Gly, was identical to the terminal of human milk bile salt-activated lipase. The human pancreatic carboxyl ester lipase has an apparent molecular weight slightly smaller than that of human milk bile salt-activated lipase (105,000 vs 125,000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, it is possible that the human pancreatic carboxyl ester lipase and human milk bile salt-activated lipase could be produced by the same gene by a different splice or post-translational modification. Alternatively, they could simply be the products of two closely related but separate genes.

Cynomolgus monkeys (Macaca fascicularis) fed monkey chow (n = 10) had a mean +/- SD post-heparin plasma lipoprotein lipase (LPL) activity level (14.7 +/- 5.5 units/ml) similar to that found in human beings (15.7 +/- 3.9 units/ml). However, the hepatic triglyceride lipase (H-TGL) in these monkeys was extremely low (0.5 +/- 0.3 units/ml) when compared with that in human beings (10.9 +/- 4.3 units/ml). The consumption of isocaloric atherogenic diets (0.2 mg cholesterol/Cal) with either saturated (P/S = 0.34) or polyunsaturated (P/S = 2.2) fat led to increased LPL activity levels (27.6 +/- 6.5 and 28.8 +/- 16.1 units/ml, respectively) and the accumulation of plasma low density lipoprotein cholesterol (LDL-C). The results indicate that cholesterol-containing atherogenic diets with either primarily saturated or polyunsaturated fat have similar potential for the increase of LPL activity. However, it is not clear whether the high dietary cholesterol content represents an obligatory component for the increase of LPL. We speculate that the high level of LPL in cynomolgus monkeys when fed a cholesterol-rich, high fat diet could be a contributing factor to the accumulation of excessive plasma LDL-C.

Mechanisms responsible for hypertriglyceridemia in Tangier disease were elucidated by an analysis of the plasma post-heparin lipolytic activities and the structural and metabolic properties of very low (VLDL) and low (LDL) density lipoproteins. The levels of lipoprotein lipase activity in six Tangier patients were significantly lower (P < 0.001) than in 40 control subjects (8.1 ± 3.3 (± S.D.) vs. 14.1 ± 3.7 units/ml). In contrast, the levels of hepatic triacylglycerol lipase were higher (P < 0.01) than in normal controls (14.4 ± 3.9 vs. 9.3 ± 4.0 units/ml). Because kinetic parameters such as Km or Vmax cannot be obtained with naturally occurring triacylglycerol-rich lipoproteins, the pseudo-first-order rate constant (k1) of triacylglycerol hydrolysis was used to assess the effectiveness of triacylglycerol-rich lipoproteins as substrates for lipoprotein lipase. The k1 values for Tangier VLDL (k1 = 0.017 ± 0.002 min−1) were significantly lower (P < 0.001) than the k1 values (0.036 ±0.008 min−1) for control VLDL. Both the Tangier and control LDL2 are similar in their resistance to the action of lipoprotein lipase, as shown by their low k1 values (0.002 ± 0.001 and 0.001 ± 0.001 mm−1, respectively). The major compositional difference between the lipoproteins of Tangier disease and normal subjects was a significant increase in the percent content of apolipoprotein A-II in all lipoprotein particles with d < 1.063 g/ml, with the greatest increase occurring in VLDL and the lowest in LDL2. These results were interpreted as indicating that, in Tangier disease, there is a lower reactivity of VLDL with lipoprotein lipase which may in part be attributed to the abnormal apolipoprotein composition. This finding, in conjunction with the reduced levels of lipoprotein lipase activity, may explain the hypertriglyceridemia in Tangier disease.

Pancreatic lipolytic enzyme activities and plasma lipids were measured in three age groups of female Fisher 344 rats (3 months [young], 12 months [adult], and 27 months [old]) in order to evaluate age-related changes and a possible correlation of these parameters. Cholesterol esterase activity was measured in pancreas homogenate, while the lipase activity was further fractionated into heparin-Sepharose unretained (lipase I) and retained (lipase II) fractions. In analogy to the lipolytic enzymes of the human pancreas, lipase I corresponds to pancreatic lipase and lipase II corresponds to pancreatic carboxylesterase. Plasma triglyceride and cholesterol levels were significantly higher in old as compared with adult and young rats. There was no significant correlation between plasma lipid and pancreatic enzyme activity levels. Cholesterol esterase and pancreatic lipase (lipase I) activity did not show any consistent change with age. Pancreatic carboxylesterase (lipase II), on the other hand, was consistently lower in adult and old animals. Although the importance of pancreatic carboxylesterase for triglyceride hydrolysis remains to be established, our results suggest that this enzyme is under long-term metabolic control. We suggest that early in life, high levels of this enzyme may be needed to supplement pancreatic lipase in order to optimize digestion of dietary fat required for optimal growth.

The incubation of human plasma very-low-density lipoprotein with human milk lipoprotein lipase results in an almost complete hydrolysis of triacylglycerols. The degradation of these substrates can be described by a consecutive reaction as follows: where k1,k2 and k3 are the apparent first-order rate constants of degradation. Using least-squares non-linear curve fitting,k1, and k2 are determined to be directly proportional to enzyme concentration. k1/k2 ratio of 1:12 is similar for both VLDL and trioleoylglycerol substrates of lipoprotein lipase. However, when trioleoylglycerol and rac-1,2-dioleoylglycerol are used as substrates, a direct measurement indicates a kl/k2 ratio of 1:1.5. This result suggests that the intermediary diacylglycerol produced by the lipoprotein reaction is incompletely re-equilibrated with the bulk of the substrate in the assay mixture. The k3, value is not proportional to lipoprotein lipase concentration, and in the enzyme concentration range studied, the value decreases when the enzyme concentration increases.

The possibility that impaired removal of lipoprotein triglyceride from the circulation may be a participating factor in the hypertriglyceridemia of the obese Zucker rat was examined. We found no significant differences in the heparin-released lipoprotein lipase (LPL) activities of the adipose tissue, skeletal muscle, and heart (expressed per gram of tissue) from the lean and obese Zucker rats. Furthermore, the kinetic properties of adipose tissue and heart LPL from the lean and obese rats were similar, indicating that the catalytic efficiency of the enzyme was unaltered in the obese animals. The postheparin plasma LPL activities of lean and obese rats were also similar. However, the postheparin plasma hepatic triglyceride lipase (H-TGL) activity in the obese rats was elevated. The higher activity of H-TGL could not alleviate the hypertriglyceridemia in these animals. Since hypertriglyceridemia in the obese rats could also be due to the hepatic production of triglyceride-rich lipoproteins which are resistant to lipolysis, we therefore isolated very low density lipoproteins (VLDL) from lean and obese rat liver perfusates and examined their degradation by highly purified human milk LPL. Although certain differences were observed in hepatic VLDL triglyceride fatty acid composition, the kinetic patterns of LPL-catalyzed triglyceride disappearance from lean and obese rat liver perfusate VLDL were similar. The isolated liver perfusate VLDL contained sufficient apolipoprotein C-II for maximum lipolysis. These results indicate that impaired lipolysis is not a contributing factor in the genesis of hypertriglyceridemia in the genetically obese Zucker rat. The hyperlipemic state may be attributed to hypersecretion of hepatic VLDL and consequent saturation of the lipolytic removal of triglyceride-rich lipoproteins from the circulation.

The effect of treatment with protein-reduced diet on plasma lipids, apolipoproteins and lipolytic activities was studied in 15 patients with chronic renal failure. Mean treatment time was 7.4 months. Before treatment serum triglycerides were elevated as were the levels of apolipoprotein C-I and especially C-III. Postheparin plasma lipolytic activities were reduced. The treatment was effective in reducing the serum urea levels but had no significant influence on either plasma lipids, apolipoprotein levels or lipolytic activities. The abnormalities of lipid transport in chronic renal failure thus seem to be more dependent on loss of renal function than the degree of uremic intoxication.