Differences in insulin action as a function of original anatomical site of newly differentiated adipocytes obtained in primary culture.

Stromal vascular cells were isolated from adipose tissue obtained from three different anatomical locations: epididymal (EPI), retroperitoneal (RP), and dorsal subcutaneous (SC), and allowed to differentiate in primary tissue culture. Cell number, protein concentration, glycerophosphate dehydrogenase, and lipoprotein lipase activity were similar in cells obtained from the EPI, RP, and SC regions, as were total insulin binding and the affinity of insulin for its receptor. However, both maximal insulin receptor tyrosine kinase activity and insulin-stimulated phosphorylation of the insulin receptor were significantly lower (P less than 0.05) in cells cultured from the SC region. In addition, newly differentiated adipocytes from the SC region were less sensitive to the ability of insulin to stimulate glucose uptake, and maximal insulin-stimulated glucose uptake by these cells was also significantly lower (P less than 0.05) when compared to cells obtained from the two other regions. Since these studies were performed on adipocyte precursor cells, allowed to differentiate to a similar degree in primary culture, the observed differences in insulin receptor phosphorylating activity, as well as the ability of insulin to stimulate glucose uptake appear to be intrinsic to adipose tissue from the three sites.

Overexpression of hormone-sensitive lipase prevents triglyceride accumulation in adipocytes.

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triglycerides within adipocytes and is thought to be the rate limiting enzyme in lipolysis; however, direct evidence to prove this concept has been lacking. The present study was designed to establish the function of HSL in adipocytes. A 2360-bp fragment containing the entire HSL coding region was cloned into the vector pCEP4 and was used to transfect the 3T3-F442A adipogenic cell line. Nondifferentiated, transfected cells were screened for HSL overexpression by indirect immunofluorescence microscopy and confirmed by immunoblotting cell extracts with anti-HSL/fusion protein antibodies and by Northern blots for HSL mRNA. Stable transfectants overexpressing HSL were obtained and cloned. Compared with undifferentiated 3T3-F442A cells transfected with pCEP4 not containing the insert (vector alone) where HSL expression was very low, undifferentiated HSL transfectants had up to a 100-fold increase in HSL activity. Likewise, immunoreactive HSL protein and HSL mRNA levels were increased up to 100-fold in HSL transfectants. When confluent cells were allowed to differentiate by exposure to insulin, HSL expression increased in vector alone transfected cells, but remained below that observed in HSL transfectants. A similar degree of differentiation was seen in both vector alone and HSL transfectants when based on the induction of lipoprotein lipase. Cellular triglyceride content increased dramatically in the vector alone transfected cells while triglyceride content was markedly reduced in the HSL transfectants. The expression of late markers of adipocyte differentiation, such as aP2 and GPDH, was diminished and appeared to vary with the degree to which HSL was overexpressed and the cellular triglyceride content was reduced. Thus, the overexpression of HSL in 3T3-F442A cells prevents differentiated adipocytes from taking on the appearance of fat cells, i.e., accumulating triglyceride. Furthermore, the overexpression of HSL directly or indirectly attenuates the expression of several genes that appear during late adipocyte differentiation.

Role of PAT proteins in lipid metabolism.

One of the central reactions in bodily energy metabolism is lipolysis in adipocytes, the reaction that governs the release of stored fatty acids from the adipocyte triacylglycerol pool, which constitutes the major energy reserve in animals. These fatty acids are then transported by serum albumin to various tissues to supply their energy requirements. This reaction was previously thought to result from phosphorylation and activation of hormone-sensitive lipase by protein kinase A (PKA) but is now known to be governed by a translocation of the lipase from the cytosol to the surface of the intracellular lipid droplet that houses the reservoir of TAG. This droplet is coated with perilipin A, which is also phosphorylated by PKA in response to lipolytic stimuli, and phosphorylation of perilipin A is essential for HSL translocation and stimulated lipolysis.

Mechanism of hypertriglyceridemia in Dahl rats.

Plasma triglyceride concentrations were shown to be higher in hypertensive (153 +/- 2 mm Hg) male Dahl salt-sensitive rats than in control Sprague-Dawley rats (122 +/- 2 mm Hg). These differences in triglyceride concentrations were seen when blood was drawn at 9 AM from unfasted animals (229 +/- 27 versus 111 +/- 8 mg/dL), at 1 PM after a 4-hour fast (186 +/- 13 versus 88 +/- 4 mg/dL), or at 9 AM after a 13-hour fast (151 +/- 6 versus 90 +/- 6 mg/dL), all p < 0.001. Total triglyceride secretion was also compared in groups of rats by determining the increment in plasma triglyceride concentration for 2 hours after blocking triglyceride removal from plasma by injecting Triton. Studies performed at 1 PM and 9 AM, after the 4- and 13-hour fast, demonstrated that total triglyceride secretion was greater (p < 0.05) in Dahl rats only when studied at 1 PM. Direct estimates of hepatic triglyceride secretion at 1 PM also demonstrated a significant (p < 0.02) increase in secretion rate by perfused livers from Dahl rats, due in part to their increased liver size. In addition, removal of prelabeled very low density lipoprotein-triglyceride in the intact rat was significantly (p < 0.05) decreased in Dahl rats. Lipoprotein lipase activity measured in skeletal muscle, heart, and adipose tissue was also significantly decreased at 9 AM and 1 PM (after 0 and 4 hours of fasting) in tissue from Dahl rats. These data confirm that Dahl rats have higher plasma triglyceride concentrations than Sprague-Dawley rats. Since both total and hepatic triglyceride secretion were somewhat greater in Dahl rats, in association with a decrease in both removal of very low density lipoprotein from plasma and decreased muscle and adipose tissue lipoprotein lipase activity, it seems likely that hypertriglyceridemia in Dahl rats results from a combination of increased triglyceride secretion and decreased triglyceride removal.

Differences in hormone-sensitive lipase expression in white adipose tissue from various anatomic locations of the rat.

Regional differences in lipolytic activity of isolated fat cells have been observed in rats, with internal fat depots displaying greater rates of lipolysis than subcutaneous fat. These differences in lipolysis have been attributed to a variety of mechanisms, including regional differences in adrenergic receptors, in adenosine sensitivity, and in regional blood flow. In the present study, we have explored whether differences in hormone-sensitive lipase (HSL), the rate-limiting enzyme in lipolysis, might contribute to regional differences in lipolysis in the rat. Adipocytes were isolated from epididymal, retroperitoneal, and dorsal-subcutaneous fat depots, and HSL activity, HSL immunoreactive protein, the rate of HSL synthesis, and the steady-state levels of HSL mRNA were assessed. HSL activity was similar in epididymal and retroperitoneal adipocytes, but was twofold to 2.5-fold greater in these cells than in subcutaneous adipocytes (P < .01). The amount of immunoreactive HSL protein was also similar in epididymal and retroperitoneal adipocytes, but was twofold greater in these cells than in subcutaneous adipocytes (P < .001). The rate of incorporation of 35S-methionine into immunoprecipitable HSL was approximately 2.5-fold greater in epididymal than in subcutaneous adipocytes (P < .01). Finally, HSL mRNA levels were similar in epididymal and retroperitoneal fat depots, and were approximately 1.4-fold greater in these cells than in subcutaneous adipocytes (P < .001). These results demonstrate that site-related differences in the rate of basal lipolysis among various fat depots in the rat are in part due to variations in the expression of HSL.

Regulation of hormone-sensitive lipase in streptozotocin-induced diabetic rats.

Insulin deficiency as seen in insulin-dependent diabetes mellitus causes an activation of lipolysis in adipose tissue that results in hydrolysis of stored triglycerides and release of large amounts of fatty acids into the plasma, leading to diabetic ketoacidosis (DKA). Hormone-sensitive lipase (HSL) is thought to be the rate-limiting enzyme of lipolysis in adipose tissue. This study was designed to examine the effects of insulin deficiency on the regulation of HSL in isolated adipocytes. Insulin deficiency was induced by a single dose of streptozotocin. After 8 days, some animals were treated with insulin, and all animals were killed 10 days after induction of insulin deficiency. Compared with levels in control rats, 10 days of insulin deficiency increased HSL activity twofold (P < .05), as assayed for neutral cholesterol esterase activity, and insulin treatment returned HSL activity to normal. HSL protein was increased twofold (P < .05) in streptozotocin-induced diabetic rats, as estimated by immunoblotting, but remained elevated after insulin treatment. Levels of HSL mRNA assessed by Northern blot analysis also increased twofold (P < .01) in adipose cells isolated from streptozotocin-induced diabetic rats, and remained elevated after insulin treatment. In conclusion, our studies suggest that 10 days of insulin deficiency increases HSL expression via pretranslational mechanisms and short-term insulin treatment returns HSL activity to normal via posttranslational mechanisms in adipose tissue.

Insulin regulates lipoprotein lipase activity in rat adipose cells via wortmannin- and rapamycin-sensitive pathways.

Lipoprotein lipase (LPL) hydrolyzes the triacylglycerol component of circulating lipoprotein particles, mediating the uptake of fatty acids into adipose tissue and muscle. Insulin is the principal factor responsible for regulating LPL activity in adipose tissue, yet the mechanisms whereby insulin controls LPL expression are unknown. The current studies used wortmannin, a specific inhibitor of phosphatidylinositol (PI) 3-kinase, and rapamycin, a specific inhibitor of activation of phosphoprotein 70 ribosomal protein S6 kinase (p70s6k), to explore some of the components of the insulin signaling pathway controlling LPL activity in adipose cells. Preincubation of isolated rat adipose cells with wortmannin completely abrogated the stimulation of LPL activity by insulin, while preincubation with rapamycin caused approximately a 60% inhibition of insulin-stimulated LPL activity. Thus, the current studies show that the regulation of adipose tissue LPL by insulin is mediated via a wortmannin-sensitive pathway, most likely PI 3-kinase, and that a rapamycin-sensitive pathway, most likely p705s6k, constitutes an important downstream component in the insulin signaling pathway through which LPL is regulated.

Acceleration by triiodothyronine of adipose conversion of rat preadipocytes from two adipose localizations.

Since we have previously reported that hyperthyroidism induces adipose tissue hyperplasia in the young rat, the effect of thyroid hormones on growth and differentiation of preadipocytes from retroperitoneal (RPAT) and epididymal (EAT) adipose tissue was studied in a primary culture system which allows a precocious cell differentiation. In this culture system, preadipocytes from RPAT exhibited a greater potentially to differentiate than cells from EAT. Chronic exposure to triiodothyronine (T3) induced an acceleration of the differentiating process as shown by a transient increase of the number of differentiated cells without alteration of cell multiplication. This effect was more important in cultures of cells from RPAT than from EAT. T3 was ineffective on lipoprotein-lipase activity but induced a stimulation of the esterification pathway which was durable and could likely be related to an increased lipid turn-over. T3 induced also a stimulation of fatty acid biosynthesis, only on the first stages of morphological differentiation which suggests that this effect could be specifically in relation with the stimulation of adipose conversion.

Depot-specific features of adipocyte progenitors revealed by primary cultures plated at low density.

Variation in growth across an animal's fat depots derives, at less in part, from inter-depot differences in pre-adipocytes. However, because of technical difficulties that have impeded the study of pre-adipocytes in primary culture, these differences are not well defined. Adipose tissue stromal cell fractions that contain pre-adipocytes grow readily in culture but invariably include cells plated at various stages of maturity. Consequently, crucial events in relatively immature cells are often obscured by activities of cells that are more mature. Therefore, one aim of the present study was to diminish the impact of cells plated near maturity by maximizing the density of immature cells. This was accomplished by the plating of stromal-vascular (SV) cell fractions at low density. Since immature pre-adipocytes proliferate much more rapidly than do mature pre-adipocytes, they become predominant. This approach made it possible for a variety of differences to be seen between cells from retroperitoneal (RP) and epididymal (Epi) tissues: Epi, but not RP, cells were seen to assemble in multi-layer clumps; multiplication was found to remain constant through at least one subculture in RP cells, but not in Epi cells; and, in a variety of ways, RP cells showed more differentiation than Epi cells. The reasons for these differences remain to be determined, but just the fact that they exist suggests that important dissimilarities among pre-adipocytes (including those responsible for nonuniform growth of fat depots) can be revealed and studied in primary cell culture.

Regulation of hormone-sensitive lipase during fasting.

Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. The activity of HSL is thought to be primarily regulated by phosphorylation-dephosphorylation reactions. Although FFA levels are elevated during fasting, it has been difficult to demonstrate an increase in HSL activity with fasting. The current studies were undertaken to explore directly the regulation of HSL expression in adipose tissue in the rat during fasting. Rats were fasted for periods up to 5 days and HSL activity, HSL immunoreactive protein, and HSL mRNA levels were measured both in intact epididymal adipose tissue and in isolated adipose cells. Fasting caused a progressive decline in total body weight and the weight of epididymal fat pads, whereas adipose cell size decreased approximately 50% after 2 days of fasting. Serum FFA levels approximately doubled within 1 day of fasting and remained elevated thereafter. Basal lipolysis, measured as glycerol release, did not increase until 2 days of fasting. HSL activity remained relatively unchanged until 3 days of fasting when it was increased twofold after 3-5 days of fasting. Likewise, HSL immunoreactive protein and HSL mRNA levels increased twofold after 3-5 days of fasting. Thus HSL activity appears to be regulated by pretranslational mechanisms during prolonged fasting. However, increases in FFA flux during short-term fasting appear to involve either post-translational control of HSL or the regulation of other enzymes.

Lipogenesis as related to endocrine control of adipose cellularity in the rat.

The incorporation rate of 3H from 3H2O in the total lipids of rat retroperitoneal adipose tissue (RPAT) according to age was followed in slightly hyperthyroid rats, in type-2 diabetic rats and in control rats. Until 12 weeks of age, the RPAT of the hyperthyroid animals exhibited hyperplasia and an increase in lipogenesis per g of wet weight or per whole fat pad. Fat cel number and lipogenesis per g of wet weight or per whole fat pad were reduced in adult diabetic rats. The concomitant changes in fat cell number and lipid synthesis in the RPAT of the two groups of treated rats have been discussed.

Effects of streptozotocin-induced diabetes on low density lipoprotein receptor expression in rat adipose tissue.

Low density lipoprotein (LDL) receptors are found in most cells, including adipose cells. LDL receptors are primarily regulated by cellular cholesterol content. Insulin and insulin deficiency have been reported to have varying effects on LDL receptors in various tissues. The present study was undertaken to assess the in vivo effects of streptozotocin-induced diabetes on LDL receptor expression and cholesterol content in adipose tissue and liver, Diabetes was induced by a single dose of streptozotocin. After 3 days, some animals were treated with insulin, and all animals were killed 10 days after induction of diabetes. Compared to control rats, 10 days of diabetes caused a decrease in adipose cell size and cellular unesterified cholesterol and cholesteryl esters, and insulin treatment returned these towards normal. No changes were observed in hepatic lipid content with diabetes or insulin treatment. Diabetes was associated with an approximately 50% reduction in immunoreactive LDL receptors in adipose cells (P < 0.01) that was returned to normal with insulin treatment. The levels of LDL receptor mRNA decreased approximately 80% (P < 0.001) in adipose cells isolated from streptozotocin-induced diabetic rats and returned to normal with insulin treatment. Hepatic LDL receptors and mRNA levels were unaffected by diabetes or insulin treatment. In conclusion, diabetes decreased LDL receptor expression in adipose cells while total cellular cholesterol content also declined.

Effect of anatomical site on insulin action and insulin receptor phosphorylation in isolated rat adipocytes.

The effects of insulin were evaluated on adipocytes isolated from three different anatomical sites in male, Sprague-Dawley rats: epididymal (EPI), retroperitoneal (RP), and dorsal subcutaneous (SC). The results indicated that maximal insulin-stimulated glucose transport was significantly lower (P less than 0.001) in cells from the SC region as compared to EPI and RP cells. In addition, the ED50 value for SC cells (259 +/- 34 pmol/l) was significantly higher than for EPI (66 +/- 5 pM) or RP adipocytes (111 +/- 32 pmol/l). Insulin inhibition of catecholamine-induced lipolysis was also significantly greater (P less than 0.001) in EPI cells as compared to RP or SC adipocytes, and that was true when expressed in absolute or relative terms. The decrease in the ability of insulin to either stimulate glucose transport or inhibit catecholamine induced lipolysis in SC cells was associated with a decrease in insulin receptor autophosphorylation and receptor tyrosine kinase activity. These data show that insulin action on isolated adipocytes varies as a function of anatomical site, and that these changes are associated with variations in insulin receptor autophosphorylation and insulin receptor tyrosine kinase activity.

Low-density lipoprotein receptors in rat adipocytes: regulation with fasting.

Adipose tissue metabolism is exquisitely sensitive to caloric intake. With increasing adiposity more triglyceride and cholesterol are stored within increasingly large adipocytes, whereas less triglyceride and cholesterol are stored as the size of the fat cell decreases. A portion of the uptake of cholesterol by adipocytes is mediated by low-density lipoprotein (LDL) receptors. The present studies addressed whether LDL receptors are differentially regulated in adipose tissue and the liver during fasting in the rat. Two days of fasting caused a reduction in body weight with an approximately 40% decrease in the epididymal fat depot and fat cell size. No changes in serum cholesterol were noted, but serum triglycerides fell approximately 55% with fasting. LDL receptors detected by immunoblotting decreased progressively with fasting to levels that were 95% below controls in adipocytes isolated from epididymal fat pads by 2-3 days. In contrast, hepatic LDL receptor expression was unaltered by fasting. After 2 days of fasting, the rate of synthesis of LDL receptors in isolated adipose cells was decreased approximately 35%, whereas levels of LDL receptor mRNA were diminished approximately 55%. It is concluded that the expression of LDL receptors in rat adipocytes is markedly downregulated during fasting through transcriptional and posttranscriptional mechanisms. Furthermore, LDL receptor expression is differentially regulated in adipose tissue and liver during fasting in the rat.

Effects of thyroid hormones on adipose tissue development in Sherman and Zucker rats.

It has been reported that mild hyperthyroidism in the young Sherman rat induces adipose tissue hyperplasia, concomitant with cell size reduction, and that hypothyroidism induces opposite effects. The present experiments were designed to study the evolution of cellularity in retroperitoneal and epididymal adipose tissue during a long term thyroxine (T4) treatment or in T4-treated rats, after the treatment had been stopped. In both cases, hyperplasia was transient with the observation that the adipocyte number observed in 3-mo-old control rats was reached earlier in T4-treated rats. In hypothyroid rats, hypoplasia was also transient, because once the treatment was stopped, the cell number overtook that of controls. Zucker rats were also treated with T4, because hypoplasia has been observed in young obese (fa/fa) rats and these rats are reported to be hypothyroid. T4 treatment increased their adipocyte number up to the level of nonobese (Fa/fa) untreated rats, while hypertrophy, although reduced, was persistent. In Sherman and Zucker rats, adipose tissue lipoprotein lipase activity was decreased by T4 treatment in parallel with and perhaps because of adipocyte size reduction. We suggest that hyperplasia induced by thyroid hormones results from a precocious differentiation of preadipocytes and does not necessarily imply an increased preadipocytes multiplication.

Hepatic and adipose tissue lipogenesis as related to age and thyroid status in the rat.

We have previously reported that, in the rat, chronic thyroxine (T4) treatment induced a transient adipose tissue hyperplasia and that, in preadipocytes cultures, lipogenesis as well as adipose conversion were enhanced by triiodothyronine. Therefore we looked for the possibility of a relationship between in vivo stimulation of adipose tissue lipogenesis and the stimulation of fat cell recruitment by thyroid hormones. Hepatic and adipose tissue de novo lipogenesis were estimated by the incorporation of 3H2O into lipids in rats of various ages made slightly hyperthyroid by daily injections of T4 (0.2 microgram/g/day) from birth. Hepatic and adipose tissue lipogenesis were increased at 3 and 6 weeks of age, no stimulation being observed when animals get older. 21 week-old animals were therefore acutely treated with 0.2 or 2 micrograms T4/g/day. In this case, only the high T4 dose was able to induce a consistent lipogenesis stimulation in liver and in retroperitoneal adipose tissue and failed to induce it in epididymal adipose tissue. These results pointed out that thyroid hormones can stimulate lipogenesis both in liver and adipose tissue. However, there is an age related fall in the sensitivity to thyroid hormones for lipogenesis stimulation, not only in the liver, but also and more pronounced in adipose tissue, in parallel to that observed in vivo for adipose differentiation; moreover, this decreased sensitivity seems to be accelerated by a long lasting hyperthyroidal state.

Alterations of lipolysis and lipoprotein lipase in chronically nicotine-treated rats.

These studies examined the cellular mechanisms for lower adiposity seen with nicotine ingestion. Rats were infused with nicotine or saline for 1 wk and adipocytes isolated from epididymal fat pads. Nicotine-infused rats gained 37% less weight and had 21% smaller fat pads. Basal lipolysis was 78% higher, whereas the maximal lipolytic response to isoproterenol was blunted in adipocytes from nicotine-infused rats. The antilipolytic actions of adenosine and the levels of serum catecholamines were unaffected by nicotine. The nicotine-induced alteration in lipolysis was not associated with any changes in hormone-sensitive lipase. Nicotine caused a 30% decrease in lipoprotein lipase (LPL) activity, without any changes in LPL mass or mRNA levels, in epididymal fat in the fed state. In contrast, LPL activity, mass, and mRNA levels in heart were increased by nicotine whether animals were fed or fasted. These studies provide evidence for multiple mechanistic events underlying nicotine-induced alterations in weight and suggest that nicotine diverts fat storage away from adipose tissue and toward utilization by muscle.

Detection of hormone-sensitive lipase in various tissues. I. Expression of an HSL/bacterial fusion protein and generation of anti-HSL antibodies.

Hormone-sensitive lipase (HSL) is an intracellular neutral lipase found in a variety of tissues, but primarily in adipose and steroidogenic tissues, that hydrolyzes triglycerides and cholesteryl esters. In the present studies, a portion of rat HSL cDNA was subcloned into a pET expression system and the resulting recombinant fusion protein was over-expressed in E. coli. The approximately 26 kD HSL/fusion protein was used to generate polyclonal antibodies in rabbits that recognize intact HSL (84 kD) in rat adipose tissue, ovary, adrenal, testis, heart, and lung, as well as in human adipose tissue. In addition, there was an approximately 89 kD protein observed in all rat tissues expressing the 84 kD protein. Unique to testes, there was an immunoreactive protein of approximately 102 kD in sexually immature rats, and additional immunoreactive proteins of approximately 113 kD and approximately 127 kD in sexually mature rats. The anti-HSL/fusion protein antibodies could remove approximately 60-80% of total neutral cholesterol esterase activity from extracts of rat adipose tissue and immunoprecipitated a single 84 kD protein after labeling of adipocytes with [32P]orthophosphate. The incorporation of 32P into the 84 kD HSL protein was stimulated 4-fold by incubation of adipocytes with isoproterenol. The half life of [35S]methionine-labeled HSL was approximately 4 h in normal rat adipocytes. The production of an HSL/fusion protein and generation of antibodies that recognize native HSL should be valuable tools in exploring the mechanisms regulating the expression of HSL activity and the function and localization of its immunoreactive proteins.

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.