Generation of antibodies against a human lipoprotein lipase fusion protein.

Antibodies generated against specific proteins are useful tools for studying the physiology and cell biology of the protein of interest. Although antibodies have been successfully generated against lipoprotein lipase (LPL) and used to elucidate many aspects of its biology, there have been problems with the specificity, affinity and availability of these antibodies. To circumvent these problems, we have expressed a portion of human LPL as a bacterial fusion protein. The human LPL bacterial fusion protein was utilized to generate polyclonal antibodies in rabbits that recognize intact human, rat and bovine LPL. Using these antibodies, it was possible to demonstrate a direct correlation between LPL mass and LPL activity from different samples of human post-heparin plasma. In addition, these antibodies were used to develop an ELISA for the measurement of LPL in tissue or plasma. This is a useful means for obtaining polyclonal antibodies to LPL in sufficient quantity and without contaminating mammalian proteins.

Transcriptional regulation of lipoprotein lipase in the heart during development in the rat.

Lipoprotein lipase (LPL) is differentially expressed in fat and heart under a variety of physiological conditions, particularly during development. LPL activity, protein and mRNA levels have been shown to rise dramatically during the first three weeks of life. The mechanism responsible for these changes in LPL in the heart during development are not fully understood. In order to address the changes in LPL in the heart during development, we measured steady state levels of LPL mRNA and the rate of LPL transcription during development. Northern blot analysis of LPL mRNA in the heart of rats from 5-120 days of age showed no changes in transcript size; however, steady state levels of LPL mRNA increased approximately 10-fold by day 20 and remained elevated thereafter. The rate of LPL transcription increased dramatically between day 5-20 and remained elevated thereafter. Thus, as opposed to other settings where posttranscriptional and posttranslational mechanisms can be important, the increased expression of LPL in rat heart that occurs during development appears to be largely due to the transcriptional activation of the gene.

Dietary regulation of the very low density lipoprotein receptor in mouse heart and fat.

The very low density lipoprotein (VLDL) receptor is a member of the LDL receptor family. As opposed to the LDL receptor, the VLDL receptor is expressed primarily in muscle and adipose tissue. Although the VLDL receptor is capable of binding lipoproteins, its functional role is still unclear. Previous studies found that VLDL receptor expression is unaffected by fasting in the rat. The current studies examined whether VLDL receptor expression is altered with fasting in the mouse. Balb/c mice were fasted for periods up to 48 hours, killed, hearts and epididymal fat obtained, and total membranes prepared. To detect the VLDL receptor a portion of the rat VLDL receptor was expressed as a bacterial fusion protein, purified and used to immunize rabbits. The antibodies raised specifically recognized intact VLDL receptor. When cardiac membranes were immunoblotted, VLDL receptor expression increased progressively with fasting, doubling at 36 hours. In contrast, VLDL receptor expression decreased progressively with fasting in membranes from epididymal fat, being reduced 70% by 48 hours. Thus, VLDL receptor expression appears to be regulated in mouse heart and fat by nutritional perturbation, supporting a potential role for the VLDL receptor in the delivery of triglycerides/fatty acids as fuel.

Detection of hormone-sensitive lipase in various tissues. II. Regulation in the rat testis by human chorionic gonadotropin.

Hormone-sensitive lipase (HSL) is an intracellular neutral lipase found in a variety of tissues, primarily in adipose and steroidogenic tissues, that hydrolyzes triglycerides and cholesteryl esters. In the rat testis steady-state levels of HSL mRNA increase dramatically during sexual maturation. In addition, HSL-like immunoreactive proteins of 84, -89, and -102 kD have been observed in sexually immature rats with additional -113 and -127 kD immunoreactive proteins expressed in mature animals. In the present studies the ability of human chorionic gonadotropin (hCG) to regulate the expression of HSL and HSL-like immunoreactive proteins in rat testes has been examined. Treatment of sexually immature rats with daily injections of hCG caused a gradual increase in HSL activity that reached an 80% rise (P < 0.01) after 5 days. This was paralleled by a 3-fold increase (P < 0.01) in the 84 kD protein representing the active HSL enzyme. The -89 kD immunoreactive protein was also increased -5-fold (P < 0.01) in parallel to the 84 kD protein and HSL activity. The -102 kD immunoreactive protein was increased by hCG treatment (P < 0.01); however, its expression did not follow changes in HSL activity or in the 84 and -89 kD immunoreactive proteins, peaking within 12 h and declining thereafter. The -113 and -127 kD immunoreactive proteins did not appear during the 5 days of hCG treatment. Steady-state levels of HSL mRNA increased 60-100% (P < 0.02) in parallel to the changes in HSL activity and in the 84 and -89 kD immunoreactive proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoproterenol decreases LDL receptor expression in rat adipose cells: activation of cyclic AMP-dependent proteolysis.

The low density lipoprotein (LDL) receptor is part of a family of proteins that mediate the uptake of lipoproteins into cells. In this paper we have demonstrated the over-expression in E. coli of a rat LDL receptor fusion protein that contains the region of the receptor sharing homology with the EGF precursor. The fusion protein was utilized to immunize rabbits and successfully generate antibodies that recognize the intact LDL receptor. These anti-LDL receptor/fusion protein antibodies were used to examine the effects of cyclic AMP on the expression of LDL receptors in isolated rat adipocytes. Incubation of adipocytes with isoproterenol caused a dose-dependent diminution in intact LDL receptors in the plasma membrane with the concomitant appearance of smaller immunoreactive proteins. Pulse-chase experiments demonstrated that isoproterenol rapidly shortened the initial half-life of intact, immunoprecipitable LDL receptors in the plasma membrane. The effects of isoproterenol on LDL receptor expression were mimicked by forskolin, by an analog of cyclic AMP, and by ACTH. In contrast, incubation with propranolol blocked the effects of isoproterenol on LDL receptor expression. While antioxidants and several different protease inhibitors had no effects, N-acetyl-leucine-leucine-methionine (ALLM) was able to prevent the isoproterenol-induced effects on LDL receptors. Thus, it appears that agents acting via cyclic AMP cause a rapid decrease in LDL receptors in the plasma membranes of isolated adipose cells due to the apparent stimulation of an ALLM-sensitive protease that degrades the LDL receptor. These results suggest a novel mechanism for the posttranscriptional regulation of LDL receptor expression in adipocytes.