Purification and characterization of hepatic triacylglycerol lipase isolated from rainbow trout,Oncorhynchus mykiss

Trialcylglycerol (TG) lipase was isolated and partially purified from rainbow trout liver. Triacylglycerol lipase activity was assayed by measuring¹⁴C-oleic acid release from¹⁴C-triolein.¹⁴C-oleic acid release was linear for up to two hours. Optimal activity occurred at pH 7.0 and 15°C. Most of the lipase activity was recovered in the cytosolic fraction. A 27,000-fold purification was achieved after Sepharose (Bio-gel A 0.5 M, 200–400 mesh) chromatography of a resuspended 20% ammonium sulfate fraction. The molecular weight of the trout hepatic lipase as determined by size-exclusion chromatography and by SDS-polyacrylamide gel electrophoresis was 40–43 kD. Lipase-mediated hydrolysis of TG resulted in the production of diacylglycerols, monoacylglycerols, and fatty acids. Kinetic analysis indicated that V_max=0.016 nmol/h/mg protein and that K_m=0.28 mM triolein. Lipolytic activity was enhanced in the presence of cAMP/ATP-Mg²⁺. These results suggest that the liver of trout possesses a neutral TG lipase that is responsible for mobilizing stored TG and is catalytically activated by phosphorylation.A part of this work was presented at the Annual Meeting of the American Society of Zoologists, December 26–30, 1990, San Antonio, TX.     

Serum lipase determination in the dog: a comparison of a titrimetric method with an automated kinetic method

An enzymatic, kinetic method for determining serum lipase activity was evaluated and compared to a standard manual method for use in dogs. The kinetic method was a commercial kit adapted for use on a tandem access clinical chemistry analyzer and utilized a series of coupled enzymatic reactions based on the hydrolysis of 1,2-diglyceride by lipase. The manual method was the Cherry-Crandall technique based on the titration of base against the acid formed by hydrolysis of an olive oil substrate by lipase. The correlation between the two methods was very good (r = 0.94). The reference range for 56 clinically healthy dogs assayed by the kinetic method was 90 to 527 U/L. Diseases associated with a greater than twofold elevation in serum lipase activity as determined by the kinetic method included pancreatitis, gastritis with liver disease, and oliguric renal failure with metabolic acidosis. In some cases, pancreatitis was seen with other clinical problems, such as gastroenteritis, diabetic ketoacidosis, duodenal mass, disseminated intravascular coagulation, and septic peritonitis. Diseases associated with serum lipase activity within the reference range or elevated less than twofold included gastritis, gastric ulcer, cholestasis, phenobarbital-induced hepatopathy, colitis, copper hepatopathy, abdominal hematoma, apocrine gland adenocarcinoma, and thrombocytopenia with pneumonia.     

Insulin induced LPL mRNA expression in THP-1 macrophage and acceleration of transferring the macrophage to foam cell

AIM:To investigate the effect of insulin on ox-LDL transferring the THP-1 cells to foam cells and influencing the LPL mRNA expression in THP-1 cells.METHODS:THP-1 cells were incubated with 50 mg/Lox-LDL and insulin at concentrations of 10 mU/L, 100 mU/L, 1 000 mU/L and 10 000 mU/L, respectively. The expression of LPL mRNA in cells was detected by RT-PCR. Lipoprotein lipase of THP-1 cells was presented by no-specific lipase staining. THP-1 cells were stained with oil red O. Accumulation of total cholesterol (TC) in THP-1 cells was determined with oxidase assay.RESULTS:In 100 mU/L、1 000 mU/L、10 000 mU/L insulin groups, LPL mRNA expression increased 2 times, the average cell perilength was longer, the percentage of positive oil red O staining cells was significant higher, the content of cholesterol in THP-1 cells was higher than in ox-LDL control (P＜0.05).CONCLUSION:Insulin accelerates transferring of THP-1 cells to foam cell with exposed to ox-LDL because LPL mRNA expression increased in the cells.     

Induction of apoptosis with Salvia miltiorrhiza monomer IH764-3 via downregulating focal adhesion kinase in H2O2-stimulated hepatic stellate cells

AIM: To investigate the effect of IH764-3 on the apoptosis of H₂O₂-stimulated hepatic stellate cells(HSCs) and the alteration of focal adhesion kinase (FAK). METHODS: The proliferation of HSCs was examined by direct cell count and the apoptosis was determined by Annexin-V/PI labeling, while the morphological change was observed by light microscopy and transmission electron microscopy. In addition, FAK mRNA was detected by RT-PCR. RESULTS: H₂O₂ promoted the proliferation of HSCs. IH764-3 induced the apoptosis of HSCs in a dose-dependent manner. The HSC apoptotic rates of different groups were 6. 35%,9. 28%,15. 10%,19. 69%,respectively, after treated with different concentrations of IH764-3 for 48 h while H₂O₂ group showed 2. 30%. In 30 mg/L group, the apoptosis rates were 6. 73%、10. 34%、15. 10% for the indicated time periods(12 h, 24 h, 48 h). In the presence of IH764-3, FAK mRNA decreased. The FAK mRNA reduction began at 2 h after adding IH764-3. CONCLUSION: IH764-3 induced the apoptosis of HSCs. Down-regulating the expression of FAK mRNA may be one of its mechanisms.     

Effect of antifibrotic herb serum on rat hepatic stellate cell proliferation and collagen synthesis

AIM: To investigate the effect of Chinese herbs, Ganxianfang(GXF), on rat hepatic stellate cells (HSC) proliferation and collagen synthesis. METHODS: Two types of herb serum, portal venous serum and circumferential venous serum, were prepared from rats infused intragastrically with 16, 8, 4 times adult dose of GXF decoction. HSC isolated from rat liver were processed with the above sera in vitro. Then we mensurated the radioactivity of HSC admixed with [[³H]H]proline and [[³H]H]thymine to judge the effect on proliferation and collagen synthesis of HSC. RESULTS: Both two types of serum collected 0.5, 1, 2 h after intragastrical infusion inhibited HSC proliferation (P＜0.05), and the serum collected 1 h after intragastrical infusion had the strongest effect (P＜0.05). Portal serum decreasea collagen synthesis (P＜0.05), but circumferential serum had no effect (P＞0.05). CONCLUSION: Inhibition of HSC proliferation and decrease of collagen synthesis may contribute to the GXF antifibrotic action.     

Effects of nitric oxide on hepatic encephalopathy in cirrhotic rats induced by LPS

AIM: To investigate the effects of nitric oxide (NO) on hepatic encephalopathy in cirrhotic rats induced by LPS. METHODS: The cirrhotic model of rats was established by complex pathogeny. Since the end of the 8 th week, the rats were intragastrically-infused with 0.9% salt, L-arginine(L-arg) and LNNA respectively for 2 weeks.The hepatic encephalopathy in cirrhotic rats were induced by 3 mg/kg LPS (ip) 4 hours before the rats were sacrificed. RESULTS: The normal behaviors and electroencephalograph were appeared in L-arg group. LNNA group showed hepatic encephalopathy. The content of NO₂^-/NO₃^- of brain tissue was markedly higher in L-arg group than LNNA group(P<0.05), but the content of histamine in brain tissue was lower in L-arg group than LNNA group(P<0.05). There was a negative correlation between the content of histamine in brain tissue and the content of NO₂^-/NO₃^- of brain tissue. CONCLUSION: NO can prevent hepatic encephalopathy in cirrhotic rats induced by LPS.