Expression of lipoprotein lipase in ovaries of the guinea pig

Guinea pig ovaries were found to have significant lipoprotein lipase (LPL) activity, corresponding to almost one-tenth the activity in paraovarian adipose tissue and in heart per gram of tissue. Northern blot analysis demonstrated the same three species of LPL mRNA in ovaries (1.8, 3.1, and 3.5 kb) as in adipose tissue. In situ hybridization showed LPL mRNA in cells of the follicular wall, and in granulosa and theca lutein cells of the mature corpus luteum. By immunolocalization, LPL was visualized in the vascular endothelium throughout the ovary, but with highest concentration in the endothelium of capillaries and large vessels of the cortical region and capillaries in the stroma of the corpus luteum. These results suggest that in the guinea pig LPL may have a function for the delivery of lipids from lipoproteins to ovarian cells.     

The relation between glycosylation and activity of guinea pig lipoprotein lipase

Previous studies have indicated that the processing of oligosaccharide chains is necessary for lipoprotein lipase to become catalytically active and may be involved in the regulation of lipase release. Guinea pig adipocytes and perfused guinea pig hearts were labeled with [35S]methionine, and lipoprotein lipase was immunoprecipitated. Digestion with endo-beta-N-acetylglucosaminidase H (Endo H) showed that the mature enzyme contains one high mannose and two complex oligosaccharide chains. Limited proteolysis indicated where in the molecule the chains are attached. Pulse-chase experiments showed that some lipase molecules were rapidly processed and appeared in the medium within 40 min. Other lipase molecules remained fully Endo H-sensitive for more than 2 h, and this form of the lipase did not appear in the medium. Both forms co-eluted with the sole lipoprotein lipase activity peak from heparin-Sepharose; this indicates that both were dimeric. Separation of the two forms was achieved by lectin chromatography and demonstrated that both were catalytically active. Cells treated with methyl-deoxynojirimycin or with deoxymannojirimycin produced and released active lipoprotein lipase which was fully Endo H-sensitive. These studies demonstrate that the trimming and processing of the oligosaccharide chains is not necessary for lipoprotein lipase to become catalytically active and be secreted, and they suggest that a comparatively large fraction of the lipase molecules is retained in the endoplasmic reticulum. Whether they ever reach the processing apparatus in the Golgi or are degraded is not clear.     

Nutritional regulation of adipose tissue lipoprotein lipase is blunted in insulin resistant rats

Background  

Hypertriglyceridemia is a common lipid abnormality accompanying insulin resistance. This study was designed to assess the contribution of dysregulation of adipose tissue lipoprotein lipase (LPL) activity to the hypertriglyceridemia in a rat model of insulin resistance.     

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) reduces lipoprotein lipase activity in the adipose tissue of the guinea pig

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) administered to young male guinea pigs at a dose of 1 microgram/kg (single intraperitoneal injection) caused a large reduction in adipose tissue lipoprotein lipase (LPL) activity. This effect occurred rapidly; a 70% decrease was noticed after 24 hour and 80% of LPL activity was lost by 48 hours when the serum triglyceride levels increased to 175% of control levels. LPL is known to play an important role in controlling the amount of free fatty acids supplied to adipose tissues. Administration of a large dose of glucose to fasted guinea pigs, which have shown a similar weight loss, but less LPL loss than TCDD-treated animals, had the effect of elevating their adipose LPL levels back to a near normal level, whereas the same treatment caused no significant increase in the LPL levels of TCDD-treated animals. Evidence indicates that the TCDD-caused decline in LPL activity is irreversible. As a consequence, the affected guinea pigs are incapable of responding to changes in nutritional status.     

Localization of lipoprotein lipase mRNA in selected rat tissues

Measurements of enzymatic activity have demonstrated that lipoprotein lipase (LPL), the principal enzyme responsible for hydrolysis of circulating triglyceride, is present in a number of tissues including brain, kidney, and adrenal gland. To determine the sites of synthesis of LPL in these tissues, in situ hybridization studies were performed using a non-sense 35S-labeled RNA probe produced from a 624-bp mouse LPL cDNA fragment. Control studies were performed with a sense RNA strand. Using 5-10-micron sections of 5-day-old rat brain, strong hybridization was found in pyramidal neurons of the hippocampus. Positive hybridization, indicating the presence of LPL mRNA, was also found in brain cortex and in the intermediate lobe of adult rat pituitary gland. Specific areas of adrenal and kidney medulla showed hybridization with the probe. LPL mRNA is, therefore, present in a number of specific regions of the body. LPL in these areas may not be important in regulating circulating levels of lipoproteins, but may be essential for cellular uptake, binding, and transfer of free fatty acids or other lipophilic substances.     

Intracellular regulation of lipoprotein lipase in human monocyte-derived macrophages

The intracellular pathway of lipoprotein lipase (LPL) has been examined in human monocyte-derived macrophages in culture. These cells were previously shown to synthesize and constitutively secrete LPL. The secretion is dependent on new enzyme synthesis. 6-d-old human monocytes have stores of mRNA for linear release of LPL up to 24 h. Enzyme activity in cells and in culture medium was almost completely inhibited by 24 h treatment with tunicamycin, an inhibitor of glycosylation. In monensin-treated cells a pronounced increase in enzyme activity was found, whereas the secreted activity was markedly reduced. This indicates that LPL in human monocytes is processed through a pH sensitive part of the Golgi complex and that the terminal glycosylation is not needed for the expression of its catalytic activity. Our results suggest that lysosomal function is not important in secretion of the enzyme, whereas vesicular transport seem to be involved in regulating LPL in human monocyte-derived macrophages in culture.     

Synthesis and regulation of lipoprotein lipase in the hippocampus

Lipoprotein lipase (LPL) expression was determined in adult rat hippocampus and compared to enzyme expression in other brain regions. Hippocampus LPL mRNA levels were at least 2.5-fold higher than those detected in the cerebral cortex, cerebellum, and remaining brain regions. Enzyme mass and activity levels in the hippocampus were also increased to a similar degree. De novo synthesis of LPL in the hippocampus was confirmed by [35S]methionine-labeling of the tissue and identification of a 57 kDa protein obtained by immunoprecipitation. Addition of an excess amount of bovine LPL completely prevented the immunoprecipitation of this protein. The effect of nutritional modulations on brain LPL activity was determined after a 12-h fast. While no significant changes were observed in other regions of the brain, hippocampus LPL activity in fasted rats increased by 60% compared to the fed control group. Simultaneously, fasting reduced adipose LPL activity by 60%. Intraperitoneal injection of ACTH over a 5-day period had no effect on hippocampus LPL activity, while adipose LPL levels increased 2.3-fold and heart LPL levels decreased 1.4-fold. We conclude that LPL is synthesized, active and regulated in a tissue-specific manner in the adult rat hippocampus.     

Expression of lipoprotein lipase associated with lung adenocarcinoma tissues

Lipoprotein lipase (LPL) plays a key role in the lipid metabolism and transporting. It can catalyze the hydrolysis of chylomicron and very low-density lipoprotein triglyceride. Moreover, the abnormality of LPL associates with many pathophysiological conditions. Herein cDNA microarray and Northern blots analysis were used to study the expression of lipoprotein lipase in lung adenocarcinoma tissues. There were 113 genes of all tested blots in cDNA microarray expressed lowly. LPL gene is expressed lowly at the average ratio 0.26 (Cy5/Cy3) in lung adenocarcinoma tissues over controls. Northern blots confirmed those changes detected from the cDNA microarray and suggested that low expression of LPL may play an important role in the lung adenocarcinoma development.     

Genomic organization of the region encoding guinea pig lipoprotein lipase; evidence for exon fusion and unconventional splicing

The coding sequence of guinea pig lipoprotein lipase (LPL) is organized into nine exons and spans a region of approximately 14 kb of the guinea pig genome. A non-conforming 5'-splice site is located on the first intron, which exhibits a 12-nucleotide perfect match with the 5'-end of the second exon. A previously described tryptic cleavage site is located on exon V, close to the 3' end of this exon. A similarity to vitellogenin resides on exons IV and V, and a putative active site is found on exon IV. A novel similarity to a fatty-acid-binding protein is noted on exon VI, adjacent to the postulated heparin-binding region. We suggest that free fatty acids (FFA) and heparin to some extent share the same site of interaction on the LPL molecule; and that a high local concentration of FFA can displace LPL from its site of action--the vascular endothelium--by competing for binding to heparan sulfate.     

Purification and characterization of lipoprotein lipase from pig myocardium.

1. Lipoprotein lipase was purified from pig myocardium by a two-step purification procedure involving (a) the formation of an enzyme-substrate complex and (b) affinity chromatography on Sepharose which contained covalently linked heparin. The purified enzyme gave in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis one main band with an apparent molecular weight of 73 000. The enzyme, which was purified 70 000-fold, had a specific activity of 860 mumol of unesterified fatty acid liberated/h per mg of protein. 2. The purified enzyme hydrolysed [14C]triolein emulsions in the absence of added cofactors but its activity was increased fivefold by adding normal human serum. Of the low-density lipoprotein apoproteins only apolipoprotein CII could be substituted for serum in activating the enzyme. This lipase had maximum activity at 0.05-0.15 M-NaCl. Heparin increased the activity of the purified enzyme twofold at low concentrations, but high concentrations inhibited. The triglyceride lipase of pig myocardium thus resembles lipoprotein lipase purified from adipose tissue and from plasma, but is clearly different from pig hepatic triglyceride lipase.     

Post-translational regulation of lipoprotein lipase activity in adipose tissue.

Changes in adipose-tissue lipoprotein lipase activity that are independent of protein synthesis were investigated in an incubation system in vitro. Under appropriate conditions at 25 degrees C a progressive increase in the enzyme activity occurs that is energy-dependent. Part of the enzyme is rapidly inactivated when the tissue is incubated with adrenaline or adrenaline plus theophylline. The mechanism of this inactivation appears to be distinct from, and to follow, the activation of the enzyme. A hypothesis is presented to account for the results in terms of an activation of the enzyme during obligatory post-translational processing and a catecholamine-regulated inactivation of the enzyme as an alternative to secretion from the adipocyte.     

Estrogens in the tissues of guinea pig embryos]

The content of estrogens was determined in tissues of the guinea pig embryos, blood of pregnant females and placenta by means of biological testing. It was the highest in embryonic ovaries at all developmental stages. The sexual dimorphism in estrogen content was found in embryonic suprarenals: it was higher in females than in males. The estrogen content in blood of male embryos and pregnant females was similar but lower than that in female embryos. Estrogens were also found in placenta and their traces were detected in spleen, brain, hypothalamus and uterus. The problem of possible participation of estrogens in the sex differentiation of female embryos is discussed.